The landscape of BRCA1 and BRCA2 large rearrangements in an international cohort of over 20 000 ovarian tumors identified using next-generation sequencing.

BACKGROUND: Approximately half of ovarian tumors have defects within the homologous recombination repair pathway. Tumors carrying pathogenic variants (PVs) in BRCA1/BRCA2 are more likely to respond to poly-ADP ribose polymerase (PARP) inhibitor treatment. Large rearrangements (LRs) are a challenging class of variants to identify and characterize in tumor specimens and may therefore be underreported. This study describes the prevalence of pathogenic BRCA1/BRCA2 LRs in ovarian tumors and discusses the importance of their identification using a comprehensive testing strategy. METHODS: Sequencing and LR analyses of BRCA1/BRCA2 were conducted in 20 692 ovarian tumors received between March 18, 2016 and February 14, 2023 for MyChoice CDx testing. MyChoice CDx uses NGS dosage analysis to detect LRs in BRCA1/BRCA2 genes using dense tiling throughout the coding regions and limited flanking regions. RESULTS: Of the 2217 PVs detected, 6.3% (N = 140) were LRs. Overall, 0.67% of tumors analyzed carried a pathogenic LR. The majority of detected LRs were deletions (89.3%), followed by complex LRs (5.7%), duplications (4.3%), and retroelement insertions (0.7%). Notably, 25% of detected LRs encompassed a single or partial single exon. This study identified 84 unique LRs, 2 samples each carried 2 unique LRs in the same gene. We identified 17 LRs that occurred in multiple samples, some of which were specific to certain ancestries. Several cases presented here illustrate the intricacies involved in characterizing LRs, particularly when multiple events occur within the same gene. CONCLUSIONS: Over 6% of PVs detected in the ovarian tumors analyzed were LRs. It is imperative for laboratories to utilize testing methodologies that will accurately detect LRs at a single exon resolution to optimize the identification of patients who may benefit from PARP inhibitor treatment.

Self-Assembling Toroidal Cell Constructs for Tissue Engineering Applications.

Developing tissues have intricate, three-dimensional (3D) organizations of cells and extracellular matrix (ECM) that provide the framework necessary to meet morphogenic and necessary demands. Migrating cells, in vivo, are exposed to numerous conflicting signals: chemokines, ECM, growth factors, and physical forces. While most of these have been studied individually in vivo or in vitro, our understanding of how cells integrate these various signals is lacking. We previously developed a novel self-organizing cellularized collagen hydrogel model that is adaptable, tunable, reproducible, and capable of mimicking the multitude of stimuli that cells experience. Our model produced self-assembled toroids of cells that were formed by 24 h. Data we present here show toroids initially form as early as 3 h after seeding. Additionally, toroids formed when cells were seeded on various collagen subtypes and were sensitive to the composition of the hydrogel. Moreover, we found differences in remodeling in toroid gels compared to gels with cells embedded in them using both a collagen binding peptide and rheology. Using scanning electron microscopy, we observed toroids forming a crater-like structure compared to whole gel contractions in mixed in gels. Finally, when multiple cells were mixed prior to seeding, heterogeneous toroids formed with some containing clusters of cells.

Plasma apolipoprotein A1 as a biomarker for Parkinson disease.

OBJECTIVE: To identify plasma-based biomarkers for Parkinson disease (PD) risk. METHODS: In a discovery cohort of 152 PD patients, plasma levels of 96 proteins were measured by multiplex immunoassay; proteins associated with age at PD onset were identified by linear regression. Findings from discovery screening were then assessed in a second cohort of 187 PD patients, using a different technique. Finally, in a third cohort of at-risk, asymptomatic individuals enrolled in the Parkinson's Associated Risk Study (PARS, n = 134), plasma levels of the top candidate biomarker were measured, and dopamine transporter (DAT) imaging was performed, to evaluate the association of plasma protein levels with dopaminergic system integrity. RESULTS: One of the best candidate protein biomarkers to emerge from discovery screening was apolipoprotein A1 (ApoA1; p = 0.001). Low levels of ApoA1 correlated with earlier PD onset, with a 26% decrease in risk of developing PD associated with each tertile increase in ApoA1 (Cox proportional hazards, p < 0.001, hazard ratio = 0.742). The association between plasma ApoA1 levels and age at PD onset was replicated in an independent cohort of PD patients (p < 0.001). Finally, in the PARS cohort of high-risk, asymptomatic subjects, lower plasma levels of ApoA1 were associated with greater putaminal DAT deficit (p = 0.037). INTERPRETATION: Lower ApoA1 levels correlate with dopaminergic system vulnerability in symptomatic PD patients and in asymptomatic individuals with physiological reductions in dopamine transporter density consistent with prodromal PD. Plasma ApoA1 may be a new biomarker for PD risk.

Osteocyte isolation and culture methods.

The aim of this paper is to present several popular methods for in vitro culture of osteocytes and osteocyte cell lines. Osteocytes are located extremely suitably within the calcified bone matrix to sense mechanical signals, and are equipped with a multitude of molecular features that allow mechanosensing. However, osteocytes are more than specialized mechanosensing cells. Several signaling molecules are preferentially produced by osteocytes, and osteocytes hold a tight reign over osteoblast and osteoclast formation and activity, but also have a role as endocrine cell, communicating with muscles or organs as remote as the kidneys. In order to facilitate further research into this fascinating cell type, three protocols will be provided in this paper. The first protocol will be on the culture of mouse (early) osteocyte cell lines, the second on the isolation and culture of primary mouse bone cells, and the third on the culture of fully embedded human osteocytes within their own three-dimensional bone matrix.

Assessment of sleepiness and unintended sleep in Parkinson's disease patients taking dopamine agonists.

OBJECTIVE: We sought to determine if patients with Parkinson's disease (PD), taking dopamine agonists (DAs) and reporting unintended sleep episodes (SEs), exhibit physiologically defined daytime sleepiness and can thus be differentiated from those taking DAs but not reporting SEs. METHODS: Twenty-four patients with abnormal Epworth Sleepiness Scale scores of >10 who were taking DAs were enrolled into one of two groups: those with SEs (SE+, n=16) and those without (SE-, n=8). Three consecutive days of testing included two nights of polysomnography followed by the Multiple Sleep Latency Test (MSLT). RESULTS: Overall frequency of pathological sleepiness (MSLT <5 min) was 42% (10/24). Mean levels of sleepiness, frequencies of pathological sleepiness, and naps with stage 2 or REM-sleep were similar between SE+ and SE- groups. Sleep tendency was similar in patients prescribed pergolide, ropinirole, and pramipexole combined with levodopa. Polysomnography testing revealed no significant differences between the groups in total sleep time, sleep efficiency, sleep architecture, or presence of restless legs syndrome or periodic leg movements. There was no relation between degree of nocturnal sleep disturbance and level of daytime sleepiness. CONCLUSIONS: The results of this study suggest SEs in PD patients occur upon a background of excessive daytime sleepiness and are unrelated to nocturnal sleep or use of a specific DA.