Monozygotic (MZ) and dizygotic (DZ) twins are often studied to determine genetic and environmental influences on complex traits, however, the biological mechanisms behind MZ and DZ twinning are not completely understood. Genomic and epigenomic studies have identified SNPs associated with DZ twinning and DNA methylation sites associated with MZ twinning. To enhance the discovery of molecular biomarkers of twinning, we compare transcriptomics and metabolomics data from MZ with those from DZ twins. We compared 42,663 RNA transcripts in 1,453 MZ twins and 1,294 DZ twins from the Netherlands Twin Register (NTR), followed by sex-stratified analyses. The 5% transcripts with lowest p-values were selected for replication analysis in 217 MZ and 158 DZ twins from the older Finnish Twin cohort (FTC). In the NTR sample, we observed upregulations of the protein coding PURG gene in MZ twins. The female-only analyses confirmed the PURG gene and identified four other genes, while the male-only analyses indicated three other genes associated with either MZ and DZ twinning. Replication results in the FTC, did not confirm PURG, but revealed seven differentially expressed genes with nominal significant p-values in both cohorts, none of which have been implicated for twinning before. Pathway enrichment showed differences in expression in the WNT-pathway and cell adhesion processes, which were previously indicated with MZ twinning though an epigenetic study, and the TGF-B pathway known to be associated with DZ twinning through genetic studies. We additionally meta-analyzed 169 serum metabolites from a NMR platform in 2,797 MZ and 2,040 DZ twins from the NTR, FTC and the FinnTwin12 (FT12), and show no metabolomic differences between the MZ and DZ twins. Overall, we identified novel transcriptomics biomarkers of twinning in peripheral blood and provide partial converging evidence for multiple pathways previously identified in the GWAS of DZ and EWAS of MZ twinning.

The authors have declared no competing interest.

the Royal Netherlands Academy of Science Professor Award (PAH/6635) to DIB and the 2023 Talent Travel Grand Award by the faculty of behaviour and movement sciences at the Vrije Universiteit Amsterdam. JvD is supported by NWO Large Scale infrastructures, Xomics (184.034.019). The NTR is supported by multiple grants from the Netherlands Organizations for Scientific Research (NOW; 480.15.001/674; 480.04.004; 400.05.717); and Medical Research (ZonMW; 912.10.020); the European Science Council (ERC) Genetics of Mental Illness (ERC Advanced, 230374); Developmental trajectories of psychopathology (NIMH 1RC2 MH089995). The metabolomics data of the NTR was collected through BBMRI: BBMRI.1 (184.021.007) and BBMRI.2 (184.033.111). The FTC has been supported by the Academy of Finland (Grants 265240, 263278, 308248, 312073, 336832 to Jaakko Kaprio and 297908 to Miina Ollikainen) and the Sigrid Juselius Foundation (to Miina Ollikainen). The transcriptome study in FTC was supported by NIH/NHLBI grant HL104125. Phenotype and genotype data collection in FinnTwin12 cohort has been supported by, ENGAGE European Network for Genetic and Genomic Epidemiology, FP7.HEALTH.F4.2007, grant agreement number 201413, National Institute of Alcohol Abuse and Alcoholism (grants AA.12502, AA.00145, and AA.09203 to R J Rose; AA15416 and K02AA018755 to D M Dick; R01AA015416 to Jessica Salvatore) and the Academy of Finland (grants 100,499, 205,585, 118,555, 141,054, 264,146, 308,248 to JK, and the Centre of Excellence in Complex Disease Genetics (grants 312,073, 336,823, and 352,792 to JKaprio).

I confirm all relevant ethical guidelines have been followed, and any necessary IRB and/or ethics committee approvals have been obtained.

Yes

The details of the IRB/oversight body that provided approval or exemption for the research described are given below:

The study protocol was approved for the NTR by the Ethical Review Board of the VU University Medical Center and informed consent was obtained from all participants. Ethical approval for all data collection waves from FT12 was obtained from the ethical committee of the Helsinki and Uusimaa University Hospital District and the Institutional Review Board of Indiana University. All data collection and sampling protocols were performed in compliance with the ethical guidelines. Parents provided consent for the twins aged 12 and 14 years old, while twins aged 17 and 22 years old provided written consent themselves for sample collection. The study protocol for the FTC was approved by the Institutional Ethics Board of the Hospital District of Helsinki and Uusimaa, Finland (ID 154/13/03/00/11) and the Institutional Review Board of Augusta University.

I confirm that all necessary patient/participant consent has been obtained and the appropriate institutional forms have been archived, and that any patient/participant/sample identifiers included were not known to anyone (e.g., hospital staff, patients or participants themselves) outside the research group so cannot be used to identify individuals.

Yes

I understand that all clinical trials and any other prospective interventional studies must be registered with an ICMJE-approved registry, such as ClinicalTrials.gov. I confirm that any such study reported in the manuscript has been registered and the trial registration ID is provided (note: if posting a prospective study registered retrospectively, please provide a statement in the trial ID field explaining why the study was not registered in advance).

Yes

I have followed all appropriate research reporting guidelines, such as any relevant EQUATOR Network research reporting checklist(s) and other pertinent material, if applicable.

Yes

The data of the Netherlands Twin Register (NTR) may be requested through the NTR data access committee (https://tweelingenregister.vu.nl/information_for_researchers/working-with-ntr-data). The Finnish Twin Cohort data used in the analysis is deposited in the Biobank of the Finnish Institute for Health and Welfare (https://thl.fi/en/web/thl-biobank/forresearchers). It is available to researchers after written application and following the relevant Finnish legislation. FinnTwin12 data analyzed in this study is not publicly available due to the restrictions of informed consent. Requests to access these datasets should be directed to the Institute for Molecular Medicine Finland (FIMM) Data Access Committee (DAC) (fimmdac@helsinki.fi) for authorized researchers who have IRB/ethics approval and an institutionally approved study plan. To ensure the protection of privacy and compliance with national data protection legislation, a data use/transfer agreement is needed, the content and specific clauses of which will depend on the nature of the requested data.

https://tweelingenregister.vu.nl/information_for_researchers/working-with-ntr-data

https://thl.fi/en/web/thl-biobank/forresearchers