O-210 DNA methylation signature of monozygotic twinning: investigating the relationship with medically assisted reproduction, higher-order gestations, and common genetic variants
Authors: Van Dongen J , Odintsova V , Hubers N , GoDMC T , Hagenbeek F , Ehli E , Hottenga J  J , De Bakker B , Henneman P , Ris-Stalpers C , Boomsma D ,   T. Twinning Genetics Consortium
Published: July 2024

Abstract

Study question
To study a DNA methylation signature of monozygotic twins in relation to medically assisted reproduction (MAR), higher-order gestations, and genome-wide common genetic variants.

Summary answer
Monozygotic twinning and MAR show largely distinct DNA methylation signatures. Monozygotic triplets, like monozygotic twins, carry a comparable but stronger DNA methylation signature.

What is known already
Monozygotic twins arise when a zygote or embryo splits during pre-implantation stages of development. Higher-order monozygotic multiples including triplets have been hypothesized to arise from multiple consecutive splitting events. The underlying mechanisms are unknown. Some studies have reported a higher rate of monozygotic twins born after the use of medically-assisted reproductive technologies. We recently identified a DNA methylation signature in blood samples from adult monozygotic twins, which showed strong replication across cohorts and in buccal samples from children. This signature consists of 834 differentially methylated locations, enriched for loci with a role in embryonic processes, cell adhesion, and metastable epi-alleles.

Study design, size, duration
We compared buccal DNA methylation profiles of 934 naturally conceived monozygotic twins to 43 monozygotic twins conceived after IVF/ICSI (mean age=9). We generated monozygotic twinning DNA methylation scores in an independent buccal dataset (59 triplets, 198 twins, and 119 siblings of twins, mean age=15). A genome-wide association study (GWAS) on the DNA methylation score was performed in two datasets from the Netherlands Twin Register (NTR) (1: Blood, N = 2841, mean age=37, 2: Buccal, N = 1150, mean age=10).

Participants/materials, setting, methods
DNA methylation was assessed with Illumina arrays (450k/EPIC). To study the relationship between DNA methylation and MAR, we performed an epigenome-wide association study (EWAS) of MAR in monozygotic twins. We applied our previously developed DNA-methylation based predictor (trained on blood methylation data from twins using elastic net regression) to novel buccal methylation data from multiples including triplets. GWAS analysis was performed in gcta using --fastGWA-mlm correcting for 10 genetic ancestry PCs and genotype platform.

Main results and the role of chance
Through EWAS, we identified 29 differentially methylated CpGs in buccal cells from monozygotic twins conceived after MAR compared to naturally conceived monozygotic twins (after Bonferroni correction), of which 8 were previously identified in an EWAS meta-analysis of IVF on cord blood from singletons at birth. None of the 834 CpGs detected in our previous EWAS meta-analysis of monozygotic twinning were associated with MAR, suggesting that they are connected to natural monozygotic twinning. The monozygotic twinning epigenetic signature showed similar performance in buccal DNA methylation data from monozygotic twins as previously observed (81% of monozygotic twins correctly classified). Sensitivity was highest for monozygotic triplets (90% correctly classified), but specificity remains moderate (66% of DZ twins correctly predicted, 53% of non-twins correctly predicted). GWAS analysis of blood 450k array data identified three genome-wide significant loci. The top SNP (rs76157694, MAF=0.25) maps to TBX4; an embryonic transcription factor primarily known for its role in hindlimb development. This finding is consistent with our previous results of MZ-differentially methylated positions being enriched within TBX4 binding motifs. GWAS analysis of the buccal EPIC array data identified no genome-wide significant loci. We are planning a GWAS meta-analysis together with the Genetics of DNA methylation consortium.

Limitations, reasons for caution
Our research provides compelling hints towards an epigenomic event in monozygotic multiples, but whether the signature represents a cause, effect, or a byproduct of monozygotic twinning remains to be established. In ongoing research, we study the signature in pre- and perinatal tissues from twins (Dutch Fetal biobank and PANDA biobank).

Wider implications of the findings
The methylation signature may serve as a biomarker to retrospectively diagnose if a person was conceived as monozygotic twin, providing novel opportunities to examine links between vanishing monozygotic twin syndrome and certain congenital disorders. Examples include Beckwith-Wiedemann Syndrome and Amyoplasia (∼10-fold higher frequency of monozygotic twins among patients).

Trial registration number
not applicable