Defining the association between trisomy 21 and placentation defects

PROJECT SUMMARY
Down’s Syndrome (T21) is the most common aneuploidy disorder in humans. T21 pregnancies are at an
increased risk of adverse pregnancy outcomes including stillbirth and intrauterine growth restriction, resulting
from placental insufficiency. T21 is also accompanied by comorbidities including congenital heart disease, which
are also linked to inadequate placentation. Coincidentally, T21 placental syncytiotrophoblasts, which act as the
primary maternal-fetal interface, fail to adequately fuse into multinucleated syncytial plaques needed for function.
We hypothesize that defects in T21 syncytiotrophoblast differentiation occur during peri-implantation
development due to dysregulation of gene expression associated with an additional copy of
chromosome 21. Ultimately, these defects likely contribute to a wide range of diseases in individuals with T21.
The long-term goals of our research are to provide a mechanism by which syncytiotrophoblast differentiation is
disrupted in T21. Our proposed work is innovative because it combines cutting edge trophoblast differentiation
protocols with functional genomics approaches. In Aim 1, we will identify transcriptional differences of T21
implantation-stage trophoblasts by differentiating six pairs of T21 and matching euploid induced pluripotent stem
(iPS) cell lines into syncytiotrophoblast-like cells. For this purpose, we will use a differentiation protocol that
includes BMP4, A83-01, and PD173074 (BAP). We will then characterize gene expression in these cell lines
using RNA-sequencing and test the functional role of differentially expressed genes in trophoblast differentiation
using knockdown and overexpression approaches. In Aim 2, we will take a hypothesis-driven approach to assess
the role of NRIP1, a transcriptional co-repressor found on chromosome 21, in trophoblast differentiation. Notably,
NRIP1 binds to estrogen receptor to suppress canonical estrogen-driven transcriptional responses. Our
preliminary data demonstrate that attenuating estrogen signaling disrupts trophoblast differentiation in a similar
manner to T21 cell lines. To better understand the role of NRIP1 in the context of Down syndrome and
trophoblast differentiation, we will knockout the third copy of NRIP1 in T21 cell lines using CRISPR/Cas9
technology. We will then measure syncytiotrophoblast function through fusion indices, gene expression, and
hormone secretion to determine whether decreasing NRIP1 in T21 lines rescues phenotypes observed in Down
syndrome. We will also overexpress a third copy of NRIP1 in euploid lines and again measure estrogen mediated
transcriptional activity and syncytiotrophoblast cell fusion to test whether increased expression of NRIP1 is
sufficient to induce T21 phenotypes. These results will clarify the involvement of NRIP1 and canonical estrogen
signaling in the T21 syncytiotrophoblast disease phenotype. Collectively, this work is significant because it will
provide a mechanistic link between placental defects and T21 pregnancies, which may inform efforts to mitigate
adverse pregnancy outcomes and comorbidities associated with Down syndrome.

Grant Number: 1R21HD118444-01
NIH Institute/Center: NIH
Principal Investigator: Justin Brumbaugh