High-load strength training compared with standard care treatment in young adults with joint hypermobility and knee pain: study protocol for a randomised controlled trial (the HIPEr-Knee study).

INTRODUCTION: Patients with generalised joint hypermobility, including knee hypermobility (GJHk), often experience knee pain and are typically managed with low-intensity strength training and/or proprioceptive training as part of standard care. However, not all patients experience satisfactory outcomes. High-load strength training may offer additional benefits, such as increased muscle cross-sectional area, neural drive and tendon stiffness, which may reduce pain and improve active knee joint stability during movement tasks and daily activities. So far, no randomised controlled trials (RCTs) have compared high-load strength training with traditional treatment strategies (standard care) for this patient group. METHODS AND ANALYSIS: In this RCT, we aim to recruit patients with GJHk and knee pain from primary care physiotherapy clinics in the Region of Southern Denmark and via social media. Patients with competing injuries or experience with high-load strength training will be excluded. Patients will be randomised (1:1 ratio) to either 2 weekly sessions of high-load strength training or standard care for 12 weeks. The primary outcome is self-reported knee pain during an activity nominated by the patient as the most aggravating for their present knee pain measured using the Visual Analogue Scale for Nominated Activity (VASNA, 0-100; 0=no pain and 100=worst imaginable pain). This will be collected at baseline, 6 weeks, 12 weeks and 12 months. Secondary outcomes include self-reported knee function and adverse events (collected at baseline, 12 weeks and 12 months), objective measurements including a 5-repetition maximum single-leg press, proprioception and single-leg-hop for distance (collected at baseline and 12 weeks), and a range of other outcome measures such as fear of movement, tendon stiffness and global perceived effect. We aim to recruit 90 patients in total to detect a 10 mm group difference in the primary outcome with 80% power. ETHICS AND DISSEMINATION: This study was funded by Independent Research Fund Denmark (grant number 2034-00088B) on 14 June 2022; the Regional Committees on Health Research Ethics for Southern Denmark approved it (S-20230050) on 30 August 2023. The first recruitment site opened on 15 February 2024, and the final results will be submitted to a peer-reviewed journal to inform rehabilitation strategies for symptomatic GJHk.Protocol version 1, dated 4 July 2024. TRIAL REGISTRATION NUMBER: NCT06277401.

Is cancer associated with frailty? Results from a longitudinal study of old Danish twins.

BACKGROUND: While age is an important risk factor for both cancer and frailty, it is unclear whether cancer itself increases the risk of frailty. We aimed to assess the association between cancer and frailty in a longitudinal cohort of older Danish twins, taking familial effect into account. METHODS: Using the Danish Cancer Registry, cancer cases were identified among participants aged 70 and over in the Longitudinal Study of Aging Danish Twins (LSADT). Frailty was evaluated over 10 years of follow-up using the frailty index (FI) and defined as FI > 0.21. Stratified Cox regressions were performed on discordant twin pairs (pairs where one twin had incident cancer and the other was cancer-free), and on all LSADT individual twins (twin pairs and singletons) with no history of cancer. RESULTS: Among the 72 discordant pairs (n=144, median age at inclusion=75) included in the study, the median FI at inclusion was 0.08 for both cancer twins and cancer-free co-twins. From the stratified Cox regression, twins with cancer had an increased hazard of developing frailty (HR=3.67, 95%CI=1.02,13.14) compared to their cancer-free co-twins. The analyses on individual twins (n=4,027) provided similar results, showing an increased hazard of frailty in individuals with cancer (HR=2.57, 95%CI= 1.77,3.74) compared to those without cancer. CONCLUSIONS: We showed a higher risk of becoming frail following a cancer diagnosis in both discordant twin pairs and individual twins. These findings support the importance of monitoring of frailty in older adults with cancer through geriatric assessments and implementation of frailty interventions.

Multi-omics association study of DNA methylation and gene expression levels and diagnoses of cardiovascular diseases in Danish Twins.

BACKGROUND: Cardiovascular diseases (CVDs) are major causes of mortality and morbidity worldwide; yet the understanding of their molecular basis is incomplete. Multi-omics studies have significant potential to uncover these mechanisms, but such studies are challenged by genetic and environmental confounding-a problem that can be effectively reduced by investigating intrapair differences in twins. Here, we linked data on all diagnoses of the circulatory system from the nationwide Danish Patient Registry (spanning 1977-2022) to a study population of 835 twins holding genome-wide DNA methylation and gene expression data. CVD diagnoses were divided into prevalent or incident cases (i.e., occurring before or after blood sample collection (2007-2011)). The diagnoses were classified into four groups: cerebrovascular diseases, coronary artery disease (CAD), arterial and other cardiovascular diseases (AOCDs), and diseases of the veins and lymphatic system. Statistical analyses were performed by linear (prevalent cases) or cox (incident cases) regression analyses at both the individual-level and twin pair-level. Significant genes (p < 0.05) in both types of biological data and at both levels were inspected by bioinformatic analyses, including gene set enrichment analysis and interaction network analysis. RESULTS: In general, more genes were found for prevalent than for incident cases, and bioinformatic analyses primarily found pathways of the immune system, signal transduction and diseases for prevalent cases, and pathways of cell-cell communication, metabolisms of proteins and RNA, gene expression, and chromatin organization groups for incident cases. This potentially reflects biology related to response to CVD (prevalent cases) and mechanisms related to regulation and development of disease (incident cases). Of specific genes, Myosin 1E was found to be central for CAD, and DEAD-Box Helicase 5 for AOCD. These genes were observed in both the prevalent and the incident analyses, potentially reflecting that their DNA methylation and gene transcription levels change both because of disease (prevalent cases) and prior disease (incident cases). CONCLUSION: We present novel biomarkers for CVD by performing multi-omics analysis in twins, hereby lowering the confounding due to shared genetics and early life environment-a study design that is surprisingly rare in the field of CVD, and where additional studies are highly needed.

FOXP3 splice variant expression in males and females in healthy populations and in kidney transplant recipients.

The forkhead box P3 (FOXP3) transcript is essential for tolerance of alloantigens. Here, we describe the expression of FOXP3 mRNA variants in healthy females and males, and in kidney transplant recipients (KTR). We measured FOXP3 in peripheral blood mononuclear cells from healthy kidney donors (N = 101), and in blood from KTRs (N = 248) before and after transplantation. FOXP3 was measured with quantitative polymerase chain reaction, and differentiated between pre-mature mRNA FOXP3, Total mature FOXP3, FOXP3 in which exon two is spliced, and full length FOXP3. We found similar levels of FOXP3 in healthy female and male kidney donors. We confirmed this result in a publicly available cohort (N = 33) of healthy individuals (GSE97475). Homogenously, female and male KTR FOXP3 levels were similar pre-transplantation, one day post-transplantation and 29 days post-transplantation. This may suggest that kidney transplantation and related immunosuppressive treatments do not influence FOXP3 expression differently in females and males. Finally, fold difference analysis revealed that KTRs express lower levels of mature FOXP3 and higher levels of pre-mature FOXP3 mRNA pre-transplant compared to healthy individuals. This finding may suggest higher pre-mRNA synthesis, lower pre-mRNA degradation, lower spliceosome efficiency or higher degradation of mature FOXP3 mRNA in kidney transplant candidates.

Popping Pills in Youth Elite Sports-Fact or Fiction? A 36-Week Prospective Cohort Study of Analgesic Use in 1195 Youth Elite Athletes and Student Controls.

OBJECTIVE: To investigate analgesic use in a cohort of Danish youth elite athletes and compare weekly analgesic use over 36 weeks to student controls. We also investigated and compared reasons for analgesic use and types of analgesics used. DESIGN: Prospective cohort study. METHODS: Six hundred ninety youth elite athletes (44% females) and 505 student controls (59% females) (aged 15-20 years) provided weekly reports on analgesic use over 36 weeks. We asked about the number of days with analgesic use, reasons for use, and types of analgesics used. Prevalence and frequency of analgesic use was compared between youth elite athletes and student controls using mixed-effects logistic regression and mixed-effects Poisson regression models. Reasons for and types of analgesics used were compared between groups using chi-square tests. Subgroup analyses were performed, stratified by sex. RESULTS: Overall, athletes had lower odds of analgesic use (odds ratio = 0.78; 95% confidence interval [CI], 0.64 to 0.95) compared with student controls. The overall usage rate was similar between the groups (incidence rate ratio = 1.04; 95% CI, 0.99 to 1.11). Subgroup analyses suggested no statistically significant differences in the odds of analgesic use. Significantly more athletes reported using analgesics to prevent or treat pain or injury in relation to sports participation and to use topical gels compared with student controls. CONCLUSION: Participating in youth elite sports was associated with lower odds of analgesic use compared to student controls, but usage rate was similar between the groups. Reasons for use and types of analgesics used differed between athletes and student controls. J Orthop Sports Phys Ther 2024;54(8):551-559. Epub 9 May 2024. doi:10.2519/jospt.2024.12407.

Association of cancer with functional decline at old age: a longitudinal study in Danish twins.

INTRODUCTION: There is evidence that older adults with cancer have a higher risk of functional decline than cancer-free older adults. However, few studies are longitudinal, and none are twin studies. Thus, we aimed to investigate the relationship between cancer and functional decline in older adult (aged 70+ years) twins. MATERIALS AND METHODS: Cancer cases in the Longitudinal Study of Aging Danish Twins were identified through the Danish Cancer Registry. Functional status was assessed using hand grip strength (6 years follow-up), and self-reported questions on mobility (10 years follow-up), and cut-offs were defined to assess functional decline. Cox regression models were performed for all the individual twins. In addition, we extended the analysis to discordant twin pairs (twin pairs with one having cancer and the other being cancer-free), to control to a certain extent for (unmeasured) shared confounders (genetic and environmental factors). RESULTS: The analysis based on individual twins showed that individual twins with cancer are at increased hazard of worsening hand grip strength (hazard ratio (HR) 1.37, 95% confidence interval (CI) 1.04, 1.80) than cancer-free twins. Among the discordant twin pairs, twins with cancer had a higher hazard of worsening hand grip strength (HR 3.50, 95% CI 1.15, 10.63) than cancer-free cotwins. In contrast, there was no evidence of a difference between the hazard of experiencing mobility decline for twins with cancer compared with cancer-free twins, in both individual twins and discordant twin pairs analyses. DISCUSSION: Cancer was associated with hand grip strength functional decline in old individual twins and discordant pairs. Our results strengthen the importance of comprehensive geriatric assessment in older adults with cancer, as well as the importance of routine assessment of functional status. Promoting physical activity through exercise training programmes could enable the prevention of functional decline in older adults with cancer.

FOXP3 full length splice variant is associated with kidney allograft tolerance.

Background: Progressive decline of allograft function leads to premature graft loss. Forkhead box P3 (FOXP3), a characteristic gene of T-regulatory cells, is known to be essential for auto-antigen tolerance. We assessed the hypothesis that low FOXP3 mRNA splice variant levels in peripheral blood cells early after transplantation are associated with progressive allograft injury. Methods: Blood samples were prospectively collected from 333 incident kidney transplant recipients on the first and 29th postoperative day. We used quantitative polymerase chain reaction to determine transcripts of 3 isotypes of FOXP3 splice variants, including pre-mature FOXP3 and full length FOXP3 (FOXP3fl). We investigated the association between FOXP3 splice variant levels and the declines in estimated glomerular filtration rate (eGFR) of more than 5ml/min/1.73m2 within the first-year post-transplant using logistic regression. Results: We observed lower FOXP3fl levels in recipients with declining eGFR (N = 132) than in recipients with stable eGFR (N = 201), (logarithmic value -4.13 [IQR -4.50 to -3.84] vs -4.00 [4.32 to -3.74], p=0.02). In ad hoc analysis pre-transplant FOXP3fl levels were similar in both groups. The association between FOXP3fl and declining eGFR was confirmed by multivariable analysis adjusted for potential confounding factors (Odds Ratio 0.51, 95% confidence interval 0.28 to 0.91: p=0.02). When stratifying FOXP3fl levels into quartiles, recipients with lower day1 FOXP3fl had the highest rate of declining eGFR (p=0.04). Conclusion: Low FOXP3fl splice variant levels at the first postoperative day in kidney transplant recipients were associated with severe decline of eGFR, a well-known surrogate for hard endpoints.

A Mass Spectrometry-Based Proteome Study of Twin Pairs Discordant for Incident Acute Myocardial Infarction within Three Years after Blood Sampling Suggests Novel Biomarkers.

Acute myocardial infarction (AMI) is a major cause of mortality and morbidity worldwide, yet biomarkers for AMI in the short- or medium-term are lacking. We apply the discordant twin pair design, reducing genetic and environmental confounding, by linking nationwide registry data on AMI diagnoses to a survey of 12,349 twins, thereby identifying 39 twin pairs (48-79 years) discordant for their first-ever AMI within three years after blood sampling. Mass spectrometry of blood plasma identified 715 proteins. Among 363 proteins with a call rate > 50%, imputation and stratified Cox regression analysis revealed seven significant proteins (FDR < 0.05): FGD6, MCAM, and PIK3CB reflected an increased level in AMI twins relative to their non-AMI co-twins (HR > 1), while LBP, IGHV3-15, C1RL, and APOC4 reflected a decreased level in AMI twins relative to their non-AMI co-twins (HR < 1). Additional 50 proteins were nominally significant (p < 0.05), and bioinformatics analyses of all 57 proteins revealed biology within hemostasis, coagulation cascades, the immune system, and the extracellular matrix. A protein-protein-interaction network revealed Fibronectin 1 as a central hub. Finally, technical validation confirmed MCAM, LBP, C1RL, and APOC3. We put forward novel biomarkers for incident AMI, a part of the proteome field where markers are surprisingly rare and where additional studies are highly needed.

Lower levels of FOXP3 are associated with prolonged inflammatory responses in kidney transplant recipients.

Background: Immunosuppressive treatment of kidney transplant recipients is mainly aimed at pro-inflammatory T effector cells, yet they also target the immunosuppressive T regulatory cells. Here, we test the hypothesis that low levels of the master gene regulator of T regulatory cells, forkhead box P3 (FOXP3) splice variants, are associated with prolonged inflammatory responses to stimuli. Methods: From blood samples obtained the first - and 29th day post-transplant, we extracted peripheral blood mononuclear cells and measured mRNA levels of Total FOXP3, pre-mature RNA FOXP3 (pre-mRNA FOXP3), full length FOXP3 (FOXP3fl) and, FOXP3 splice variant excluding exon two (FOXP3d2). We defined the primary outcome as the number of days in which C reactive protein (CRP) was above 50 mg/L. CRP levels were gathered in two periods, the first from the second to 29 days post-transplant, and the second from 30 to 57 days post-transplant. The association was tested using adjusted negative binomial regression. Results: From 507 included kidney transplant recipients, 382 recipients had at least one CRP measurement >50 mg/L in the first period, median duration of elevated CRP was 4 days [interquartile range (IQR) 2 to 6]. In the second period, 69 recipients had at least one CRP measurement >50 mg/L, median duration of elevated CRP was 3 days [IQR 2 to 5]. In the first period, we found a significant association between lower levels of Total FOXP3 and prolonged duration of CRP elevation, incidence rate ratio 0.61 (95% confidence interval 0.46-0.80), p<0.01. Conclusion: Lower levels of total FOXP3 mRNA levels in peripheral blood of kidney transplant recipients are associated with prolonged duration of inflammatory responses regardless of the underlying stimuli.

Identifying genetic variants regulating MGMT gene expression - A study in monozygotic Danish twins.

Identifying genetic factors affecting the regulation of the O-6-Methylguanine-DNA Methyltransferase (MGMT) gene and estimating the genetic contribution of the MGMT gene through within-pair correlation in monozygotic twin pairs is of particular importance in various types of cancer such as glioblastoma. We used gene expression data in whole blood from 448 monozygotic twins from the Middle Age Danish Twins (MADT) study to investigate genetic regulation of the MGMT gene by performing a genome-wide association study (GWAS) of the variation in MGMT expression. Additionally, we estimated within-pair dependence measures of the expression values looking for the genetic influence of significant identified genes. We identified 243 single nucleotide polymorphisms (SNPs) significantly (p < 5e-8) associated with expression of MGMT, all located on chromosome 10 near the MGMT gene. Of the 243 SNPs, 7 are novel cis-eQTLs. By further looking into the suggestively significant SNPs (increasing cutoff to p = 1e-6), we identified 11 suggestive trans-eQTLs located on chromosome 17. These variants were in or proximal to a total of seven genes, which may regulate MGMT expression. The within-pair correlation of the expression of MGMT, TRIM37, and SEPT4 provided the upper bound genetic influence of these genes. Overall, identifying cis- or trans-acting genetic variations regulating the MGMT gene can pave the way for a better understanding of the MGMT gene function and ultimately in understanding the patient's sensitivity to therapeutic alkylating agents.

Predictive value of geriatric oncology screening and geriatric assessment of older patients with cancer: A randomized clinical trial protocol (PROGNOSIS-RCT).

INTRODUCTION: Comprehensive geriatric assessment (CGA) has been shown to reduce frailty in older patients in general. In older patients with cancer, frailty affects quality of life (QoL), physical function, and survival. However, few studies have examined the effect of CGA as an additional intervention to antineoplastic treatment. This protocol presents a randomized controlled trial, which aims to evaluate the effects of CGA-based interventions in older patients with cancer and Geriatric 8 (G8) identified frailty. MATERIALS AND METHODS: This randomized controlled trial will include patients, age 70+ years, with solid malignancies and G8 frailty (G8 ≤ 14). Patients will be separated into two groups, with different primary endpoints, depending on palliative or curative antineoplastic treatment initiation, and subsequently randomized 1:1 to either CGA with corresponding interventions or standard of care, along with standardized antineoplastic treatment. A geriatrician led CGA with corresponding interventions and clinical follow-up will be conducted within one month of antineoplastic treatment initiation. The interdisciplinary CGA will cover multiple geriatric domains and employ a standard set of validated assessment tools. Primary endpoints will be physical decline measured with the 30-s Chair-Stand-Test at three months (palliative setting) and unplanned hospital admissions at six months (curative setting). Additional outcomes include QoL, treatment toxicity and adherence, occurrence of polypharmacy, potential drug interactions, potential inappropriate medications, and survival. The primary outcomes will be analyzed using a mixed model regression analysis (30-s chair stand test) and linear regression models (unplanned hospitalizations), with an intention to treat approach. Power calculations reveal the need to enroll 134 (palliative) and 188 (curative) patients. DISCUSSION: The present study will examine whether CGA, as an additional intervention to antineoplastic treatment, can improve endpoints valued by older patients with cancer. Inclusion began November 2020 and is ongoing, with 37 and 29 patients recruited April 15th, 2021. Registration:NCT04686851.

Global Gene Expression Profiling and Transcription Factor Network Analysis of Cognitive Aging in Monozygotic Twins.

Cognitive aging is one of the major problems worldwide, especially as people get older. This study aimed to perform global gene expression profiling of cognitive function to identify associated genes and pathways and a novel transcriptional regulatory network analysis to identify important regulons. We performed single transcript analysis on 400 monozygotic twins using an assumption-free generalized correlation coefficient (GCC), linear mixed-effect model (LME) and kinship model and identified six probes (one significant at the standard FDR < 0.05 while the other results were suggestive with 0.18 ≤ FDR ≤ 0.28). We combined the GCC and linear model results to cover diverse patterns of relationships, and meaningful and novel genes like APOBEC3G, H6PD, SLC45A1, GRIN3B, and PDE4D were detected. Our exploratory study showed the downregulation of all these genes with increasing cognitive function or vice versa except the SLC45A1 gene, which was upregulated with increasing cognitive function. Linear models found only H6PD and SLC45A1, the other genes were captured by GCC. Significant functional pathways (FDR < 3.95e-10) such as focal adhesion, ribosome, cysteine and methionine metabolism, Huntington's disease, eukaryotic translation elongation, nervous system development, influenza infection, metabolism of RNA, and cell cycle were identified. A total of five regulons (FDR< 1.3e-4) were enriched in a transcriptional regulatory analysis in which CTCF and REST were activated and SP3, SRF, and XBP1 were repressed regulons. The genome-wide transcription analysis using both assumption-free GCC and linear models identified important genes and biological pathways implicated in cognitive performance, cognitive aging, and neurological diseases. Also, the regulatory network analysis revealed significant activated and repressed regulons on cognitive function.

Predictive Value of Geriatric Oncology Screening and Geriatric Assessment in Older Patients with Solid Cancers: Protocol for a Danish prospective cohort study (PROGNOSIS-G8).

INTRODUCTION: Older patients with cancer constitute a heterogeneous group with varying degrees of frailty; therefore, geriatric assessment with initial geriatric oncology screening is recommended. The Geriatric 8 (G8) and the modified Geriatric 8 (mG8) are promising screening tools with high accuracy and an association with survival. However, evidence is sparse regarding patient-centered outcomes. This protocol describes a study, which aims to address the predictive and prognostic value of the G8 and mG8, with quality of life (QoL) as the primary outcome. MATERIALS AND METHODS: In this single-center prospective cohort study, patients, age ≥70 years with solid malignancies, will be screened with the G8 and mG8 prior to receiving 1st line antineoplastic treatment. Patients will contribute medical record data including; cancer type, Charlson comorbidity index score, performance status, and treatment intent, type, and dosage, at baseline. Patients will complete QoL questionnaires (EORTC QLQ-C30 and ELD-14) at baseline, 3, 6, 9, and 12-months follow-up. Two functional measurements (the 30-s chair stand test and the handgrip strength test) will be conducted at baseline to assess the added predictive and prognostic value. At 12 months follow-up, initially administered treatment and treatment adherence will be recorded and assessed with generalized linear models, while overall survival and cancer-specific survival will be assessed using survival analysis models with time-varying covariates. The relationship between frailty (G8 ≤ 14, mG8 ≥ 6) and QoL within 12 months will be examined using mixed regression models. DISCUSSION: Geriatric oncology screening may identify a subgroup of older patients with frailty, at risk of experiencing diminishing QoL and poor treatment adherence. With the proposed screening program, patients who require treatment modification and additional support to maintain their QoL may be identified. It is our hope, that these insights may facilitate the formation of national guidelines for the treatment of older patients with cancer. Registration:NCT04644874.

Differential lncRNA expression profiling of cognitive function in middle and old aged monozygotic twins using generalized association analysis.

Cognitive impairment is the most prominent symptom in neurodegenerative disorders affecting quality of life and mortality. However, despite years of research, the molecular mechanism underlying the regulation of cognitive function and its impairment is poorly understood. This study aims to elucidate the role of long non-coding RNAs (lncRNAs) expression and lncRNA-mRNA interaction networks, by analyzing lncRNA expression in whole blood samples of 400 middle and old aged monozygotic twins in association with cognitive function using both linear models and a generalized correlation coefficient (GCC) to capture the diverse patterns of correlation. We detected 13 probes (p < 1e-03) displaying nonlinear and 7 probes (p < 1e-03) showing linear correlations. After combining the results, we identified 20 lncRNA probes with p < 1e-03. The top lncRNA probes were annotated to genes, along with the non-coding MALAT1, that play roles in neurodegenerative diseases. The top lncRNAs were linked to functional clusters including peptidyl-glycine modification, vascular smooth muscle cells, mitotic spindle organization and protein tyrosine phosphatase. In addition, mapping of the top significant lncRNAs to the lncRNA-mRNA interaction network detected significantly enriched biological pathways involving neuroactive ligand-receptor interaction, proteasome and chemokines. We show that GCC served as a complementary approach in detecting lncRNAs missed by the conventional linear models. A combination of GCC and linear models identified lncRNAs of diverse patterns of association enriched for GO biological and molecular functions meaningful in cognitive performance and cognitive decline. The novel lncRNA regulatory network further contributed to detect significant pathways implicated in cognition.

Genetic and environmental determinants of O6-methylguanine DNA-methyltransferase (MGMT) gene methylation: a 10-year longitudinal study of Danish twins.

BACKGROUND: Epigenetic inactivation of O6-methylguanine DNA-methyltransferase (MGMT) is associated with increased sensitivity to alkylating chemotherapeutic agents in glioblastoma patients. The genetic background underlying MGMT gene methylation may explain individual differences in treatment response and provide a clue to a personalized treatment strategy. Making use of the longitudinal twin design, we aimed, for the first time, to estimate the genetic contributions to MGMT methylation in a Danish twin cohort. METHODS: DNA-methylation from whole blood (18 monozygotic (MZ) and 25 dizygotic (DZ) twin pairs) repeated 10 years apart from the Longitudinal Study of Aging Danish Twins (LSADT) were used to search for genetic and environmental contributions to DNA-methylation at 170 CpG sites of across the MGMT gene. Both univariate and bivariate twin models were applied. The intraclass correlations, performed on cross-sectional data (246 MZ twin pairs) from an independent study population, the Middle-Aged Danish Twins (MADT), were used to assess the genetic influence at each CpG site of MGMT for replication. RESULTS: Univariate twin model revealed twelve CpG sites showing significantly high heritability at intake (wave 1, h2 > 0.43), and seven CpG sites with significant heritability estimates at end of follow-up (wave 2, h2 > 0.5). There were six significant CpG sites, located at the gene body region, that overlapped among the two waves (h2 > 0.5), of which five remained significant in the bivariate twin model, which was applied to both waves. Within MZ pair correlation in these six CpGs from MADT demarks top level of genetic influence. There were 11 CpGs constantly have substantial common environmental component over the 10 years. CONCLUSIONS: We have identified 6 CpG sites linked to the MGMT gene with strong and persistent genetic control based on their DNA methylation levels. The genetic basis of MGMT gene methylation could help to explain individual differences in glioblastoma treatment response and most importantly, provide references for mapping the methylation Quantitative Trait Loci (meQTL) underlying the genetic regulation.

Novel DNA methylation marker discovery by assumption-free genome-wide association analysis of cognitive function in twins.

Privileged by rapid increase in available epigenomic data, epigenome-wide association studies (EWAS) are to make a profound contribution to understand the molecular mechanism of DNA methylation in cognitive aging. Current statistical methods used in EWAS are dominated by models based on multiple assumptions, for example, linear relationship between molecular profiles and phenotype, normal distribution for the methylation data and phenotype. In this study, we applied an assumption-free method, the generalized correlation coefficient (GCC), and compare it to linear models, namely the linear mixed model and kinship model. We use DNA methylation associated with a cognitive score in 400 and 206 twins as discovery and replication samples respectively. DNA methylation associated with cognitive function using GCC, linear mixed model, and kinship model, identified 65 CpGs (p < 1e-04) from discovery sample displaying both nonlinear and linear correlations. Replication analysis successfully replicated 9 of these top CpGs. When combining results of GCC and linear models to cover diverse patterns of relationships, we identified genes like KLHDC4, PAPSS2, and MRPS18B as well as pathways including focal adhesion, axon guidance, and some neurological signaling. Genomic region-based analysis found 15 methylated regions harboring 11 genes, with three verified in gene expression analysis, also the 11 genes were related to top functional clusters including neurohypophyseal hormone and maternal aggressive behaviors. The GCC approach detects valuable methylation sites missed by traditional linear models. A combination of methylation markers from GCC and linear models enriched biological pathways sensible in neurological function that could implicate cognitive performance and cognitive aging.

EWASex: an efficient R-package to predict sex in epigenome-wide association studies.

SUMMARY: Epigenome-Wide Association Study (EWAS) has become a powerful approach to identify epigenetic variations associated with diseases or health traits. Sex is an important variable to include in EWAS to ensure unbiased data processing and statistical analysis. We introduce the R-package EWASex, which allows for fast and highly accurate sex-estimation using DNA methylation data on a small set of CpG sites located on the X-chromosome under stable X-chromosome inactivation in females. RESULTS: We demonstrate that EWASex outperforms the current state of the art tools by using different EWAS datasets. With EWASex, we offer an efficient way to predict and to verify sex that can be easily implemented in any EWAS using blood samples or even other tissue types. It comes with pre-trained weights to work without prior sex labels and without requiring access to RAW data, which is a necessity for all currently available methods. AVAILABILITY AND IMPLEMENTATION: The EWASex R-package along with tutorials, documentation and source code are available at https://github.com/Silver-Hawk/EWASex. SUPPLEMENTARY INFORMATION: Supplementary data are available at Bioinformatics online.

Generalized correlation coefficient for genome-wide association analysis of cognitive ability in twins.

Despite a strong genetic background in cognitive function only a limited number of single nucleotide polymorphisms (SNPs) have been found in genome-wide association studies (GWASs). We hypothesize that this is partially due to mis-specified modeling concerning phenotype distribution as well as the relationship between SNP dosage and the level of the phenotype. To overcome these issues, we introduced an assumption-free method based on generalized correlation coefficient (GCC) in a GWAS of cognitive function in Danish and Chinese twins to compare its performance with traditional linear models. The GCC-based GWAS identified two significant SNPs in Danish samples (rs71419535, p = 1.47e-08; rs905838, p = 1.69e-08) and two significant SNPs in Chinese samples (rs2292999, p = 9.27e-10; rs17019635, p = 2.50e-09). In contrast, linear models failed to detect any genome-wide significant SNPs. The number of top significant genes overlapping between the two samples in the GCC-based GWAS was higher than when applying linear models. The GCC model identified significant genetic variants missed by conventional linear models, with more replicated genes and biological pathways related to cognitive function. Moreover, the GCC-based GWAS was robust in handling correlated samples like twin pairs. GCC is a useful statistical method for GWAS that complements traditional linear models for capturing genetic effects beyond the additive assumption.

Network based analysis of microarray gene expression profiles in response to electroacupuncture.

Electroacupuncture (EA) has been extensively considered as a tool for treating diseases and relieving various pains. However, understanding the molecular mechanisms underlying its effect is of high importance. In this study, we performed a weighted gene co-expression network analysis (WGCNA) on data collected from a microarray experiment to investigate the relationship underlying EA within three factors, time, frequency and tissue regions (periaqueductal grey (PAG) and spinal dorsal horn (DH)) as well as the biological implication of gene expression changes. Gene expression on rats in PAG-DH regions induced by EA with 2 Hz and 100 Hz at l h and 24 h were measured using microarray technology. The WGCNA was performed to identify distinct network modules related to EA effects. To find the biological function of genes and pathways, the gene ontology (GO) Consortium was applied and the gene-gene interaction network of top genes in important modules was visualized. We identified one network module (466 genes) which is significantly associated with time, another module (402 genes) significantly related to frequency, and three modules each consisting of 1144, 402 and 3148 genes that are significantly associated with tissue regions. Furthermore, meaningful biological pathways were enriched in association with each of the experimental factors during EA stimulation. Our analysis showed the robustness of WGCNA and revealed important genes within specific modules and pathways which might be activated in response to EA analgesia. The findings may help to clarify the underlying mechanisms of EA and provide references for future verification of this study.

Differential long noncoding RNA profiling of BMI in twins.

Aim: Many efforts have been deployed to identify genetic variants associated with BMI. Alternatively, we explore epigenetic contribution to BMI variation by focusing on long noncoding RNAs (lncRNAs) which represents a key layer of epigenetic control. Materials & methods: We analyzed lncRNA expression in whole blood of 229 monozygotic twin pairs in association with BMI using generalized estimating equations. Results & conclusion: Six lncRNA probes were identified as significant (false discovery rate <0.05), with BMI showing causal effects on the expression of the significant lncRNAs. Functional annotation of differential profiles identified Gene ontology biological processes including kidney development, regulations of lipid biosynthetic process, circadian rhythm, notch signaling, etc. Whole blood lncRNAs are significantly expressed in response to BMI variation.