Overview. The primary epigenome-wide association study (EWAS) of left-handedness was performed in two cohorts with DNA methylation data in whole blood (Illumina, 450k): NTR adults57 (N = 2,682 individuals including twins, mean age at methylation 36.5, standard deviation (SD) 12.7), and ALSPAC adults58,59 (N = 1,232, mean age at methylation 48.98, SD 5.55). EWAS analyses were performed in each dataset separately, and summary statistics were combined in the meta-analysis (N = 3,914) testing 409,563 CpGs. As this is a meta-analysis of existing DNA methylation datasets, no analyses were done to pre-determine sample sizes, but the sample size is larger compared to previously published DNA methylation studies of handedness41,42. We tested whether the EWAS signal was enriched in nearby loci detected in the previous GWAS on handedness13. We carried out within-pair twin analysis in MZ twins discordant for handedness (Nadults= 133 twin pairs, Nchildren= 86 twin pairs). Secondary analyses were performed in different tissues: in cord blood and peripheral blood in ALSPAC offspring60, i.e. the children of ALSPAC participants that contributed to the primary EWAS (N = 791 with DNA methylation data at birth, at 7, and 17 years old (Illumina 450k chip), and/or at 24 years old (Illumina EPIC array)), and in buccal cells from an independent group of children from the NTR61,62 (N = 946 twins, mean age 9.5, SD 1.85, Illumina EPIC array). We performed EWAS analyses in each dataset and examined correlations between the regression coefficients of top CpGs (ranked on ascending p-value) from each EWAS analysis. Finally, we created and tested polygenic and DNA methylation scores for left-handedness. The study method and design are presented in Fig. 1 and Fig. 3. Detailed cohort information is provided in Appendix 1.
Handedness. NTR. Information on hand preference for adults and children was collected by surveys and in small subgroups from laboratory-based projects. Parental reports on children were collected at 5 years and included seven items for different activities, from which the item “What hand does child use for drawing?” was selected. The four answer categories were left-handed, right-handed, both hands and do not know. Multiple adult surveys included the question: “Are you right-handed or left-handed? (4 surveys)” or “Are you predominantly left-handed or right-handed?” (3 surveys). The three answer categories were left-handed, right-handed, and both. For a small number of adult self-reports at younger ages (14, 16 or 18) or parental assessment at age 5 were also available.
ALSPAC. Adults (mothers and fathers) were asked which hand they used to write, draw, throw, hold a racket or bat, brush their teeth, cut with a knife, hammer a nail, strike a match, rub out a mark, deal from a pack of cards or thread a needle (11 questions). Child handedness was assessed at 42 months by questionnaire in which the mother was asked which hand the child used to draw, throw a ball, color, hold a toothbrush, cut with a knife, and hit things (6 questions). Responses were scored − 1, 0 or 1 for left, either or right, respectively. Handedness was coded as 1 for left-handed or 0 for right-handed in both cohorts.
DNA methylation and genotyping. DNA methylation was measured with the Infinium HumanMethylation450 BeadChip Kit which measures more than 450,000 methylation sites (primary analysis in adults in NTR and ALSPAC and secondary analysis in ALSPAC offspring at birth, 7 and 17 years old), or the and Infinium MethylationEPIC BeadChip which measures more than 850,000 methylation sites (secondary analysis in ALSPAC offspring at 24 years old and NTR children). Genotyping for polygenic risk scores was done on multiple platforms with imputation of the target data using reference haplotypes from 1000 genomes reference panel. Cohort-specific details on biosample collection, DNA methylation profiling, quality control, cell-type proportions measurements and genotyping are described in Appendix 1.
Intergroup differences. We tested if there were differences in characteristics that were included in EWAS models (such as age at biological sample collection, sex, body mass index (BMI), smoking status at blood collection for adults, and gestational age, maternal smoking during pregnancy, birth weight for children, cell proportions/percentages in buccal swabs and in blood samples) between left- and right-handed individuals by generalized estimating equations (GEE) to accommodate the relatedness among the twins in NTR, and by standard logistic regression in ALSPAC. The R package ‘gee’ was used with the following specifications: binomial (for ordinal data) link function, 100 iterations, and the ‘exchangeable’ option to account for the correlation structure within families and within persons. Right- and lefthanded MZ discordant twins were compared with paired t-test for the traits that were not identical in twins (birth weight, BMI, smoking, cell percentages). All statistical tests here and below were two-tailed.
Epigenome-Wide Association Analyses. Primary analyses. The association between DNA methylation levels and left-handedness was tested for each site under a linear model (ALSPAC) or generalized estimating equation (GEE) model accounting for relatedness of twins (NTR). DNA methylation beta-values were the dependent variable and were typically normally distributed. The following predictors were included in the basic model: handedness (coded as 0 = right-handed and 1 = left-handed), sex, age at blood sampling, percentage of blood cells for blood samples, and technical covariates in NTR and ALSPAC (see Appendix 1). An adjusted model was fitted to account for BMI and smoking status at blood draw in both NTR and ALSPAC adult cohorts, because BMI and smoking have large effects on DNA methylation in adults38,46. The primary results reported in the paper are based on the fully adjusted model. The models are described in Appendix 2. Throughout the text, we refer to regression coefficients from the EWAS, which represent the methylation difference between left-handed and right-handed individuals on the methylation beta-value scale. A positive regression coefficient (β) means a higher methylation level in left-handed individuals. The value of an individual on the methylation scale is commonly also symbolized as beta (β) and ranges from 0 to 1, where 0 represents a methylation level of 0% and 1 represents a methylation level of 100%.
Secondary analyses. The same basic models were fitted to the data from ALSPAC and NTR children. For DNA methylation in buccal cells, cell proportions (epithelial cells, natural killer cells) for buccal samples were included instead of percentage of blood cells. As several characteristics, such as gestational age and birthweight, affect DNA methylation48,63, we included these in the adjusted model in children (see Appendix 2).
In the within-pair analysis of discordant MZ twins, paired t-tests were employed to test for methylation differences between the left-handed and the right-handed twins. Paired t-tests were performed in R on residual methylation levels, which were obtained by adjusting the DNA methylation β-values for sample plate, array row, cell proportions in buccal samples in children and sample plate, array row, and percentages of blood cells in adults. Additional covariates, birth weight in children and BMI and smoking status in adults, were added in adjusted model. Age, sex, maternal smoking, and gestational age were not included because these variables are identical in MZ twins.
To account for multiple testing, we considered Bonferroni correction and a False Discovery Rate (FDR) of 5%. The Bonferroni corrected p-value threshold was calculated by dividing 0.05 by the number of genome-wide CpGs tested, and false discovery rate (FDR) q-values were computed with the R package ‘qvalue’ with default settings. The Bayesian inflation factor (λ) was calculated with the R package Bacon64 (see Supplementary Table 5).
Meta-analysis. A meta-analysis was performed in METAL65 based on estimates (regression coefficients) and standard errors from the EWAS of handedness performed with GEE in NTR and linear regression in ALSPAC. NTR and ALSPAC adult cohorts were combined. In total, 409,563 CpG sites present in both cohorts were tested with statistical significance evaluated after Bonferroni correction and at an FDR q-value < 0.05.
Comparison of top CpGs from different analyses. To compare top CpGs from different analyses, we repeated the NTR EWAS analyses in adults in children and meta-analysis with discordant MZ twin pairs removed to avoid sample overlap. We selected methylation sites that overlapped in 13 analyses with adjusted model (meta-analysis, meta-analysis without discordant MZ twins, EWAS NTR adults, EWAS NTR adults without discordant MZ twins, EWAS ALSPAC adults, EWASs ALSPAC at birth, 7, 17, 24 years, EWAS NTR children, EWAS NTR children without discordant twins, and within-pair analyses of discordant MZ twin adults and children) that resulted in 379,924 methylation sites. We calculated Pearson correlations for effect estimates of the top 100 CpGs ranked by p-value from one analysis with the effect estimates of the same CpGs in other analyses. Statistical significance of correlations was assessed after Bonferroni correction for the number of correlations tested: α = 0.05/(13 × 13–13) = 0.0003.
Differentially methylated regions. We used the R dmrff library36 for R to identify regions where CpG sites showed evidence for association with handedness. Dmrff identifies DMRs by meta-analysing EWAS summary statistics from CpG sites in each region while adjusting for dependencies between the sites and uncertainty in the EWAS effects (https://github.com/perishky/dmrff). In this study, dmrff was applied separately in the NTR and ALSPAC cohorts, and then used to identify DMRs in common between the cohorts by meta-analysis. As previously described36, DMR meta-analysis preceded by first identifying candidate DMRs using the EWAS meta-analysis summary statistics, calculating DMR statistics for these candidates in each cohort separately, and then meta-analysing the DMR statistics across the two cohorts. The DMR effect size is a weighted sum of the EWAS effects for each CpG site (i.e. methylation differences between LH and RH). All dmrff p-values were adjusted for multiple tests (Bonferroni adjustment) by multiplying them by the total number of DMRs considered. We report significant regions (Padj < 0.05) including at least two CpG sites within a 500bp window observed to be nominally associated with handedness by EWAS (P < 0.05). The average absolute DNA methylation difference in the region between left-handers and right-handers is calculated as the sum of absolute regression coefficients of each CpG in the region divided by the number of CpGs. We plotted the DMRs with the coMET R Bioconductor package66 to graphically display additional information on physical location of CpGs, correlation between sites, statistical significance, and functional annotation (annotation tracks included genes Ensembl, CpG islands (UCSC), regulation Ensembl).
GWAS follow-up. GWAS follow-up analyses were performed to examine whether CpGs within a 1 Mb window of loci detected by the GWAS for left-handedness13, on average, showed a stronger association with left-handedness than other genome-wide methylation sites (Infinium HumanMethylation450 BeadChip). We obtained a SNP list based on the GWAS meta-analysis without NTR, ALSPAC, and 23andMe by Cuellar-Partida et al.13 (196,419 individuals, NSNPs = 13,550,404), from which we selected all SNPs with a p-value < 1.0 ×10− 08, < 1.0 ×10− 06, and < 1.0×10− 05, and determined the distance of each Illumina 450k methylation site to each SNP. To test whether methylation sites near GWAS loci were more strongly associated with left-handedness, meta-analysis EWAS test statistics were regressed on a variable indicating if the CpG is located within a 1 Mb window from SNPs associated with handedness (1 = yes, 0 = no):
|Zscore| = Intercept + 𝛽category x * Category x,
where |Zscore| represents the absolute Zscore for a CpG from the EWAS meta-analysis of handedness; 𝛽category x represents the estimate for category x, i.e. the change in the EWAS test statistic associated with a one-unit change in category x (e.g. being within 1 Mb of SNPs associated with left-handedness). For each enrichment test, bootstrap standard errors were computed with 2000 bootstraps with the R-package “simpleboot”. Statistical significance was assessed at α = 0.05. As control analysis, the same follow-up was performed using GWAS summary statistics on a trait that is unrelated to handedness – type 2 diabetes in UK Biobank cohort (N = 244,890) 43. GWAS summary statistics were downloaded from GWASAtlas (https://atlas.ctglab.nl/traitDB/3686; 41204_E11_logistic.EUR.sumstats.MACfilt.txt; accessed on February 1 2021).
We looked up CpG sites associated with handedness-associated SNPs with a p-value < 1.0 ×10− 08 (based on the GWAS meta-analysis by Cuellar-Partida et al.13 without 23andMe, NTR and ALSPAC, resulting in a list of 420 SNPs) using the mQTL database maintained by the Genetics of DNA Methylation Consortium (GoDMC, N = 27,750 European samples; http://mqtldb.godmc.org.uk/about)44. We then checked if associations with handedness were observed for these sites in our EWAS meta-analysis and DMR meta-analysis.
EWAS follow-up. To examine previously reported associations for epigenome-wide significant DMRs associated with left-handedness in our study, we looked up CpGs from the regions in the EWAS Atlas67 (https://bigd.big.ac.cn/ewas/tools; accessed on August 1 2020) and EWAS catalogue68 (http://www.ewascatalog.org; access on November 1 2020).
Polygenic and methylation scores. Polygenic scores (PGS) for handedness were calculated based on the GWAS meta-analysis without 23andMe by Cuellar-Partida et al.13. To avoid overlap between the discovery and target samples, summary statistics without NTR and ALSPAC were requested (196,419 individuals, NSNPs = 13,550,404). The linkage disequilibrium (LD) weighted betas were calculated using a LD pruning window of 250 KB, with the fraction of causal SNPs set at 0.50 by LDpred69. We randomly selected 2500 2nd degree unrelated individuals from each cohort as a reference population to calculate the LD patterns. The resulting betas were used to calculate the PGSs in each dataset using the PLINK 1.9 software. All PGSs were standardized (mean of 0 and standard deviation of 1). Methylation scores (MS) were calculated in NTR based on EWAS summary statistics obtained from ALSPAC, and vice versa, as previously done to create methylation scores for BMI and height70. We calculated same-tissue same-age DNA-methylation scores based on methylation data from NTR adults (blood) and ALSPAC parents (blood), and cross-tissue DNA-methylation scores based on data from NTR and ALSPAC offspring, with DNA methylation measured in buccal cells, and blood, respectively (see Fig. 1). For each individual, a weighted score sum was calculated for left-handedness by multiplying the methylation value at a given CpG by the effect size of the CpG (β), and then summing these values over all CpGs: DNA methylation score (i) = β1*CpG1i + β2*CpG2i … + βn*CpGni, where CpGn is the methylation level at CpG site n in participant i, and βn is the regression coefficient at CpGn taken from summary statistics of the EWAS analysis. All methylation scores were standardized (mean of 0 and standard deviation of 1). We used weights from summary statistics of EWASs in four cohorts: NTR adults, ALSPAC adults, NTR children, ALSPAC offspring at 7 years old. Subsets of CpGs to be included in methylation scores were selected based on p-value < 1⋅10− 1, < 1⋅10− 3, and < 1⋅10− 5. We analysed the predictive value of the left-handedness polygenic scores and methylation scores in NTR and ALSPAC adult and child cohorts from our EWAS study. To quantify the variance explained by the PGS and MS, we used the approach proposed by Lee et al.71, where coefficients of determination (R2) for binary responses are calculated on the liability scale. The equations of all models are provided in Appendix 2. Statistical significance was assessed following Bonferroni correction for the number of scores tested (PGS and 3 MSs). This resulted in α = 0.05/4 = 0.0125, nominal significance at 0.05.
Ethics statement. All methods were performed in accordance with the Declaration of Helsinki. For NTR, the study was approved by the Central Ethics Committee on Research Involving Human Subjects of the VU University Medical Centre, Amsterdam, an Institutional Review Board certified by the U.S. Office of Human Research Protections (IRB number IRB00002991 under Federal-wide Assurance FWA00017598; IRB/institute codes, NTR 03-180). All subjects provided written informed consent. For children, written informed consent was given by their parents. For ALSPAC, ethical approval for the study was obtained from the ALSPAC Ethics and Law Committee and the Local Research Ethics Committees. All subjects provided written informed consent. For children, written informed consent was given by their mothers.
Data availability. The HumanMethylation450 BeadChip data from the NTR are available as part of the Biobank-based Integrative Omics Studies (BIOS) Consortium in the European Genome-phenome Archive (EGA), under the accession code EGAD00010000887 (https://ega-archive.org/datasets/EGAD00010000887). The Infinium MethylationEPIC from NTR are available from the Netherlands Twin Register on reasonable request (https://tweelingenregister.vu.nl/information_for_researchers/working-with-ntr-data). DNA methylation data from ALSPAC are available at ALSPAC and can be provided on request. The study website contains details of all the data that is available through a fully searchable data dictionary and variable search tool (http://www.bristol.ac.uk/alspac/researchers/our-data). The code used to perform the primary and secondary analyses is available at https://github.com/MRCIEU/handedness-ewas. The pipeline for the DNA methylation array analysis developed by the Biobank-based Integrative Omics Study (BIOS) consortium are available here: https://molepi.github.io/DNAmArray_workflow/. EWAS summary statistics for the top 100 CpGs are given in Supplemental Tables 6-11 and 15-29. The full EWAS summary statistics from the meta-analysis with basic and adjusted model are provided in Supplemental Tables 32 and 33. The full summary statistics for all other analyses are available upon request from the corresponding author.