The first clinical validation of whole-genome screening on standard trophectoderm biopsies of preimplantation embryos.

Objective: To validate the performance of our laboratory-developed whole-genome screening assay within clinical preimplantation genetic testing environments.

Design: Perform a laboratory-developed whole-genome assay on both cell lines and trophectoderm biopsies, subsequently employing the next-generation sequencing procedure to reach a sequencing depth of 30X. Adhere to the Genome Analysis Toolkit best practices for accuracy, sensitivity, specificity, and precision calculations by comparing samples with references.

The molecular basis of Rac-GTP action-promoting binding of p67 to Nox2 by disengaging the β hairpin from downstream residues.

p67 fulfils a key role in the assembly/activation of the NADPH oxidase by direct interaction with Nox2. We proposed that Rac-GTP serves both as a carrier of p67 to the membrane and an inducer of a conformational change enhancing its affinity for Nox2. This study provides evidence for the latter function: (i) oxidase activation was inhibited by p67 peptides (106-120) and (181-195), corresponding to the β hairpin and to a downstream region engaged in intramolecular bonds with the β hairpin, respectively; (ii) deletion of residues 181-193 and point mutations Q115R or K181E resulted in selective binding of p67 to Nox2 peptide (369-383); (iii) both deletion and point mutations led to a change in p67 , expressed in increased apparent molecular weights; (iv) p67 was bound to p67 peptide (181-195) and to a cluster of peptides (residues 97-117), supporting the participation of selected residues within these sequences in intramolecular bonds; (v) p67 failed to bind to Nox2 peptide (369-383), following interaction with Rac1-GTP, but a (p67 -Rac1-GTP) chimera exhibited marked binding to the peptide, similar to that of p67 deletion and point mutants; and (vi) size exclusion chromatography of the chimera revealed its partition in monomeric and polymeric forms, with binding to Nox2 peptide (369-383) restricted to polymers.

p67 -derived self-assembled peptides prevent Nox2 NADPH oxidase activation by an auto-inhibitory mechanism.

Activation of the Nox2-dependent NADPH oxidase is the result of a conformational change in Nox2 induced by interaction with the cytosolic component p67 . In preliminary work we identified a cluster of overlapping 15-mer synthetic peptides, corresponding to p67 residues 259-279, which inhibited oxidase activity in an in vitro, cell-free assay, but the results did not point to a competitive mechanism. We recently identified an auto-inhibitory intramolecular bond in p67 , one extremity of which was located within the 259-279 sequence, and we hypothesized that inhibition by exogenous peptides might mimic intrinsic auto-inhibition.

p67 binds to a newly identified site in Nox2 following the disengagement of an intramolecular bond-Canaan sighted?

Activation of the phagocyte NADPH oxidase involves a conformational change in Nox2. The effector in this process is p67 and there is evidence for a change in the configuration of p67 being required for binding to Nox2. To study this, we measured binding of p67 to a library of Nox2 peptides and binding of NusA-Nox2 fusion proteins to p67 .

Utility and First Clinical Application of Screening Embryos for Polygenic Disease Risk Reduction.

For over 2 decades preimplantation genetic testing (PGT) has been in clinical use to reduce the risk of miscarriage and genetic disease in patients with advanced maternal age and risk of transmitting disease. Recently developed methods of genome-wide genotyping and machine learning algorithms now offer the ability to genotype embryos for polygenic disease risk with accuracy equivalent to adults. In addition, contemporary studies on adults indicate the ability to predict polygenic disorders with risk equivalent to monogenic disorders.