Publications

Many studies using reporter assays have demonstrated that 3' untranslated regions (3'-UTRs) regulate gene expression by controlling mRNA stability and translation. Due to intrinsic limitations of heterologous reporter assays, we sought to develop a gene editing approach to investigate the regulatory activity of 3'-UTRs in their native context. We initially used dual-CRISPR (clustered, regularly interspaced, short palindromic repeats)-Cas9 targeting to delete DNA regions corresponding to nine chemokine 3'-UTRs that destabilized mRNA in a reporter assay. Targeting six chemokine 3'-UTRs increased chemokine mRNA levels as expected. However, targeting CXCL1, CXCL6 and CXCL8 3'-UTRs unexpectedly led to substantial mRNA decreases. Metabolic labeling assays showed that targeting these three 3'-UTRs increased mRNA stability, as predicted by the reporter assay, while also markedly decreasing transcription, demonstrating an unexpected role for 3'-UTR sequences in transcriptional regulation. We further show that CRISPR-Cas9 targeting of specific 3'-UTR elements can be used for modulating gene expression and for highly parallel localization of active 3'-UTR elements in the native context. Our work demonstrates the duality and complexity of 3'-UTR sequences in regulation of gene expression and provides a useful approach for modulating gene expression and for functional annotation of 3'-UTRs in the native context.

OBJECTIVE

To describe the natural history and perinatal outcomes of monochorionic diamniotic twins with midtrimester isolated oligohydramnios (iOligo).

MATERIALS AND METHODS

We performed a retrospective study of iOligo patients who were initially referred for the management of evolving twin-twin transfusion syndrome (TTTS) or selective intrauterine growth restriction (sIUGR). iOligo was defined as a maximum vertical pocket of amniotic fluid of ≤2 cm in the iOligo twin's sac and normal fluid level (>2 and <8 cm) in the co-twin's sac. "Group A" patients did not subsequently develop TTTS or sIUGR Type II (umbilical artery persistent absent or reversed end-diastolic flow), and "Group B" patients did develop TTTS or sIUGR Type II. Results are reported as median (range).

RESULTS

Of the 828 patients with complicated monochorionic twin gestations referred for possible TTTS or sIUGR, 36 (4.3%) were initially diagnosed with iOligo. After initial consultation, two patients terminated and one was lost to follow-up, resulting in a final study population of 33. Group A had 10 patients (30.3%) and Group B had 23 patients (69.7%). In Group A, nine of the 10 were expectantly managed, resulting in a median gestational age (GA) at delivery of 34.7 (18.0-36.4) weeks, a 30-day perinatal survival of at-least-one twin of 88.9% (8/9), and dual 30-day survivors in 8/9 (88.9%). In Group B, 12 (52.2%) developed TTTS and 11 (47.8%) developed sIUGR Type II. Fifteen Group B patients had laser surgery, resulting in a median GA at delivery of 33.7 (26.4-37.1) weeks, a 30-day perinatal survival of at-least-one twin of 100% (15/15), and dual survivorship of 46.7% (7/15).

CONCLUSIONS

Our findings show that the majority of patients with midtrimester iOligo have fetal growth restriction of the affected twin and subsequently progress to TTTS or sIUGR Type II.