Tracing the timing of monozygotic twinning using somatic mutations.

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Project summary In this proposal, we aim to prove a hypothesis on the timing of monozygotic twinning that was first proposed in the 1950s. The hypothesis states that the monozygotic twinning occurs at different stages during embryogenesis, and that the timing can be inferred by the sharing of chorion/amnion at birth. While this simple and intuitive hypothesis was incorporated into many textbooks, the scientific proof of the hypothesis is lacking. The advent of whole-genome sequencing now allows us to use embryonic somatic mutations as molecular barcodes of cellular lineages in early embryogenesis. We will use these somatic mutations to trace the embryonic history of monozygotic twins with different degrees of chorionicity/amnionicity. We have shown that detection of such embryonic somatic mutations is indeed possible on sequencing platforms. With our bioinformatics pipeline, we hope to prove the hypothesis that the timing of monozygotic twinning is related to the status of chorions/amnions shared by the monozygotic twins. In Aim 1, we will sequence twin families to identify embryonic somatic mutations in monozygotic twins with varying degrees of chorionicity/amnionicity as well as dizygotic twins. We will compare and validate the different pattern of embryonic somatic mutations in different types of twins. In Aim 2, we will create a simulation model of the twinning event to understand the genomic differences between the twins. This will allow us to estimate the timing of twinning with different types of chorionicity/amnionicity. In Aim3, we will trace the twinning event at the single cell level by isolating single cells from each twin to draw the cellular lineage trees of the twins. Our study will emphasize the discordance between monozygotic twins and prove the commonly accepted hypothesis that the chorionicity/amnionicity of monozygotic twins is correlated with the timing of the splitting event of monozygotic twins. This will be the first study to comprehensively characterize the discordance of monozygotic twins from a genomics perspective using embryonic somatic mutations. We will also show that these embryonic somatic mutations can explain some of the discordance seen in monozygotic twins, and that not all discordance in the twins should be referred to as non-genetic etiology. Tracing the embryonic somatic mutations at the single cell level will help us precisely time the twinning event. Therefore, this study will enhance our understanding of the basic biology of twinning and add another mechanism of explanation to genetic discordance in monozygotic twins, which may also affect treatment and counselling options for the monozygotic twins.