Validation of a non-invasive prenatal test for fetal RhD, C, c, E, K and Fya antigens.

We developed and validated a next generation sequencing-(NGS) based NIPT assay using quantitative counting template (QCT) technology to detect RhD, C, c, E, K (Kell), and Fya (Duffy) fetal antigen genotypes from maternal blood samples in the ethnically diverse U.S. population. Quantitative counting template (QCT) technology is utilized to enable quantification and detection of paternally derived fetal antigen alleles in cell-free DNA with high sensitivity and specificity. In an analytical validation, fetal antigen status was determined for 1061 preclinical samples with a sensitivity of 100% (95% CI 99-100%) and specificity of 100% (95% CI 99-100%). Independent analysis of two duplicate plasma samples was conducted for 1683 clinical samples, demonstrating precision of 99.9%. Importantly, in clinical practice the no-results rate was 0% for 711 RhD-negative non-alloimmunized pregnant people and 0.1% for 769 alloimmunized pregnancies. In a clinical validation, NIPT results were 100% concordant with corresponding neonatal antigen genotype/serology for 23 RhD-negative pregnant individuals and 93 antigen evaluations in 30 alloimmunized pregnancies. Overall, this NGS-based fetal antigen NIPT assay had high performance that was comparable to invasive diagnostic assays in a validation study of a diverse U.S. population as early as 10 weeks of gestation, without the need for a sample from the biological partner. These results suggest that NGS-based fetal antigen NIPT may identify more fetuses at risk for hemolytic disease than current clinical practice, which relies on paternal genotyping and invasive diagnostics and therefore is limited by adherence rates and incorrect results due to non-paternity. Clinical adoption of NIPT for the detection of fetal antigens for both alloimmunized and RhD-negative non-alloimmunized pregnant individuals may streamline care and reduce unnecessary treatment, monitoring, and patient anxiety.

ReporterSeq reveals genome-wide dynamic modulators of the heat shock response across diverse stressors.

Understanding cellular stress response pathways is challenging because of the complexity of regulatory mechanisms and response dynamics, which can vary with both time and the type of stress. We developed a reverse genetic method called ReporterSeq to comprehensively identify genes regulating a stress-induced transcription factor under multiple conditions in a time-resolved manner. ReporterSeq links RNA-encoded barcode levels to pathway-specific output under genetic perturbations, allowing pooled pathway activity measurements via DNA sequencing alone and without cell enrichment or single-cell isolation. We used ReporterSeq to identify regulators of the heat shock response (HSR), a conserved, poorly understood transcriptional program that protects cells from proteotoxicity and is misregulated in disease. Genome-wide HSR regulation in budding yeast was assessed across 15 stress conditions, uncovering novel stress-specific, time-specific, and constitutive regulators. ReporterSeq can assess the genetic regulators of any transcriptional pathway with the scale of pooled genetic screens and the precision of pathway-specific readouts.

Quantification of Hsp90 availability reveals differential coupling to the heat shock response.

The heat shock response (HSR) is a protective gene expression program that is activated by conditions that cause proteotoxic stress. While it has been suggested that the availability of free chaperones regulates the HSR, chaperone availability and the HSR have never been precisely quantified in tandem under stress conditions. Thus, how the availability of chaperones changes in stress conditions and the extent to which these changes drive the HSR are unknown. In this study, we quantified Hsp90 chaperone availability and the HSR under multiple stressors. We show that Hsp90-dependent and -independent pathways both regulate the HSR, and the contribution of each pathway varies greatly depending on the stressor. Moreover, stressors that regulate the HSR independently of Hsp90 availability do so through the Hsp70 chaperone. Thus, the HSR responds to diverse defects in protein quality by monitoring the state of multiple chaperone systems independently.

ATP hydrolysis by RAD50 protein switches MRE11 enzyme from endonuclease to exonuclease.

MRE11-RAD50 is a key early response protein for processing DNA ends of broken chromosomes for repair, yet how RAD50 nucleotide dynamics regulate MRE11 nuclease activity is poorly understood. We report here that ATP binding and ATP hydrolysis cause a striking butterfly-like opening and closing of the RAD50 subunits, and each structural state has a dramatic functional effect on MRE11. RAD50-MRE11 has an extended conformation in solution when MRE11 is an active nuclease. However, ATP binding to RAD50 induces a closed conformation, and in this state MRE11 is an endonuclease. ATP hydrolysis opens the RAD50-MRE11 complex, and MRE11 maintains exonuclease activity. Thus, ATP hydrolysis is a molecular switch that converts MRE11 from an endonuclease to an exonuclease. We propose a testable model in which the open-closed transitions are used by RAD50-MRE11 to discriminate among DNA ends and drive the choice of recombination pathways.