BRCA1 secondary splice-site mutations drive exon-skipping and PARP inhibitor resistance.

BRCA1 splice isoforms Δ11 and Δ11q can contribute to PARP inhibitor (PARPi) resistance by splicing-out the mutation-containing exon, producing truncated, partially-functional proteins. However, the clinical impact and underlying drivers of BRCA1 exon skipping remain undetermined. We analyzed nine ovarian and breast cancer patient derived xenografts (PDX) with BRCA1 exon 11 frameshift mutations for exon skipping and therapy response, including a matched PDX pair derived from a patient pre- and post-chemotherapy/PARPi. BRCA1 exon 11 skipping was elevated in PARPi resistant PDX tumors. Two independent PDX models acquired secondary BRCA1 splice site mutations (SSMs), predicted in silico to drive exon skipping. Predictions were confirmed using qRT-PCR, RNA sequencing, western blots and BRCA1 minigene modelling. SSMs were also enriched in post-PARPi ovarian cancer patient cohorts from the ARIEL2 and ARIEL4 clinical trials. We demonstrate that SSMs drive BRCA1 exon 11 skipping and PARPi resistance, and should be clinically monitored, along with frame-restoring secondary mutations.

Understanding the emergence of the boson peak in molecular glasses.

A common feature of glasses is the "boson peak", observed as an excess in the heat capacity over the crystal or as an additional peak in the terahertz vibrational spectrum. The microscopic origins of this peak are not well understood; the emergence of locally ordered structures has been put forward as a possible candidate. Here, we show that depolarised Raman scattering in liquids consisting of highly symmetric molecules can be used to isolate the boson peak, allowing its detailed observation from the liquid into the glass. The boson peak in the vibrational spectrum matches the excess heat capacity. As the boson peak intensifies on cooling, wide-angle x-ray scattering shows the simultaneous appearance of a pre-peak due to molecular clusters consisting of circa 20 molecules. Atomistic molecular dynamics simulations indicate that these are caused by over-coordinated molecules. These findings represent an essential step toward our understanding of the physics of vitrification.

Rapid genome sequencing identifies a novel de novo SNAP25 variant for neonatal congenital myasthenic syndrome.

Congenital myasthenic syndrome (CMS) is a group of 32 disorders involving genetic dysfunction at the neuromuscular junction resulting in skeletal muscle weakness that worsens with physical activity. Precise diagnosis and molecular subtype identification are critical for treatment as medication for one subtype may exacerbate disease in another (Engel et al., Lancet Neurol 14: 420 [2015]; Finsterer, Orphanet J Rare Dis 14: 57 [2019]; Prior and Ghosh, J Child Neurol 36: 610 [2021]). The SNAP25-related CMS subtype (congenital myasthenic syndrome 18, CMS18; MIM #616330) is a rare disorder characterized by muscle fatigability, delayed psychomotor development, and ataxia. Herein, we performed rapid whole-genome sequencing (rWGS) on a critically ill newborn leading to the discovery of an unreported pathogenic de novo SNAP25 c.529C > T; p.Gln177Ter variant. In this report, we present a novel case of CMS18 with complex neonatal consequence. This discovery offers unique insight into the extent of phenotypic severity in CMS18, expands the reported SNAP25 variant phenotype, and paves a foundation for personalized management for CMS18.

Epithelial-to-Mesenchymal Transition Supports Ovarian Carcinosarcoma Tumorigenesis and Confers Sensitivity to Microtubule Targeting with Eribulin.

Ovarian carcinosarcoma (OCS) is an aggressive and rare tumor type with limited treatment options. OCS is hypothesized to develop via the combination theory, with a single progenitor resulting in carcinomatous and sarcomatous components, or alternatively via the conversion theory, with the sarcomatous component developing from the carcinomatous component through epithelial-to-mesenchymal transition (EMT). In this study, we analyzed DNA variants from isolated carcinoma and sarcoma components to show that OCS from 18 women is monoclonal. RNA sequencing indicated that the carcinoma components were more mesenchymal when compared with pure epithelial ovarian carcinomas, supporting the conversion theory and suggesting that EMT is important in the formation of these tumors. Preclinical OCS models were used to test the efficacy of microtubule-targeting drugs, including eribulin, which has previously been shown to reverse EMT characteristics in breast cancers and induce differentiation in sarcomas. Vinorelbine and eribulin more effectively inhibited OCS growth than standard-of-care platinum-based chemotherapy, and treatment with eribulin reduced mesenchymal characteristics and N-MYC expression in OCS patient-derived xenografts. Eribulin treatment resulted in an accumulation of intracellular cholesterol in OCS cells, which triggered a downregulation of the mevalonate pathway and prevented further cholesterol biosynthesis. Finally, eribulin increased expression of genes related to immune activation and increased the intratumoral accumulation of CD8+ T cells, supporting exploration of immunotherapy combinations in the clinic. Together, these data indicate that EMT plays a key role in OCS tumorigenesis and support the conversion theory for OCS histogenesis. Targeting EMT using eribulin could help improve OCS patient outcomes. SIGNIFICANCE: Genomic analyses and preclinical models of ovarian carcinosarcoma support the conversion theory for disease development and indicate that microtubule inhibitors could be used to suppress EMT and stimulate antitumor immunity.

Comprehensive variant calling from whole-genome sequencing identifies a complex inversion that disrupts ZFPM2 in familial congenital diaphragmatic hernia.

BACKGROUND: Genetic disorders contribute to significant morbidity and mortality in critically ill newborns. Despite advances in genome sequencing technologies, a majority of neonatal cases remain unsolved. Complex structural variants (SVs) often elude conventional genome sequencing variant calling pipelines and will explain a portion of these unsolved cases. METHODS: As part of the Utah NeoSeq project, we used a research-based, rapid whole-genome sequencing (WGS) protocol to investigate the genomic etiology for a newborn with a left-sided congenital diaphragmatic hernia (CDH) and cardiac malformations, whose mother also had a history of CDH and atrial septal defect. RESULTS: Using both a novel, alignment-free and traditional alignment-based variant callers, we identified a maternally inherited complex SV on chromosome 8, consisting of an inversion flanked by deletions. This complex inversion, further confirmed using orthogonal molecular techniques, disrupts the ZFPM2 gene, which is associated with both CDH and various congenital heart defects. CONCLUSIONS: Our results demonstrate that complex structural events, which often are unidentifiable or not reported by clinically validated testing procedures, can be discovered and accurately characterized with conventional, short-read sequencing and underscore the utility of WGS as a first-line diagnostic tool.

TSPAN6 is a suppressor of Ras-driven cancer.

Oncogenic mutations in the small GTPase RAS contribute to ~30% of human cancers. In a Drosophila genetic screen, we identified novel and evolutionary conserved cancer genes that affect Ras-driven tumorigenesis and metastasis in Drosophila including confirmation of the tetraspanin Tsp29Fb. However, it was not known whether the mammalian Tsp29Fb orthologue, TSPAN6, has any role in RAS-driven human epithelial tumors. Here we show that TSPAN6 suppressed tumor growth and metastatic dissemination of human RAS activating mutant pancreatic cancer xenografts. Whole-body knockout as well as tumor cell autonomous inactivation using floxed alleles of Tspan6 in mice enhanced KrasG12D-driven lung tumor initiation and malignant progression. Mechanistically, TSPAN6 binds to the EGFR and blocks EGFR-induced RAS activation. Moreover, we show that inactivation of TSPAN6 induces an epithelial-to-mesenchymal transition and inhibits cell migration in vitro and in vivo. Finally, low TSPAN6 expression correlates with poor prognosis of patients with lung and pancreatic cancers with mesenchymal morphology. Our results uncover TSPAN6 as a novel tumor suppressor receptor that controls epithelial cell identify and restrains RAS-driven epithelial cancer.

The Extracellular Milieu of Toxoplasma's Lytic Cycle Drives Lab Adaptation, Primarily by Transcriptional Reprogramming.

Evolve and resequencing (E&R) was applied to lab adaptation of Toxoplasma gondii for over 1,500 generations with the goal of mapping host-independent in vitro virulence traits. Phenotypic assessments of steps across the lytic cycle revealed that only traits needed in the extracellular milieu evolved. Nonsynonymous single-nucleotide polymorphisms (SNPs) in only one gene, a P4 flippase, fixated across two different evolving populations, whereas dramatic changes in the transcriptional signature of extracellular parasites were identified. Newly developed computational tools correlated phenotypes evolving at different rates with specific transcriptomic changes. A set of 300 phenotype-associated genes was mapped, of which nearly 50% is annotated as hypothetical. Validation of a select number of genes by knockouts confirmed their role in lab adaptation and highlights novel mechanisms underlying in vitro virulence traits. Further analyses of differentially expressed genes revealed the development of a "pro-tachyzoite" profile as well as the upregulation of the fatty acid biosynthesis (FASII) pathway. The latter aligned with the P4 flippase SNP and aligned with a low abundance of medium-chain fatty acids at low passage, indicating this is a limiting factor in extracellular parasites. In addition, partial overlap with the bradyzoite differentiation transcriptome in extracellular parasites indicated that stress pathways are involved in both situations. This was reflected in the partial overlap between the assembled ApiAP2 and Myb transcription factor network underlying the adapting extracellular state with the bradyzoite differentiation program. Overall, E&R is a new genomic tool successfully applied to map the development of polygenic traits underlying in vitro virulence of T. gondii. IMPORTANCE It has been well established that prolonged in vitro cultivation of Toxoplasma gondii augments progression of the lytic cycle. This lab adaptation results in increased capacities to divide, migrate, and survive outside a host cell, all of which are considered host-independent virulence factors. However, the mechanistic basis underlying these enhanced virulence features is unknown. Here, E&R was utilized to empirically characterize the phenotypic, genomic, and transcriptomic changes in the non-lab-adapted strain, GT1, during 2.5 years of lab adaptation. This identified the shutdown of stage differentiation and upregulation of lipid biosynthetic pathways as the key processes being modulated. Furthermore, lab adaptation was primarily driven by transcriptional reprogramming, which rejected the starting hypothesis that genetic mutations would drive lab adaptation. Overall, the work empirically shows that lab adaptation augments T. gondii's in vitro virulence by transcriptional reprogramming and that E&R is a powerful new tool to map multigenic traits.

Novel temporal and spatial patterns of metastatic colonization from breast cancer rapid-autopsy tumor biopsies.

BACKGROUND: Metastatic breast cancer is a deadly disease with a low 5-year survival rate. Tracking metastatic spread in living patients is difficult and thus poorly understood. METHODS: Via rapid autopsy, we have collected 30 tumor samples over 3 timepoints and across 8 organs from a triple-negative metastatic breast cancer patient. The large number of sites sampled, together with deep whole-genome sequencing and advanced computational analysis, allowed us to comprehensively reconstruct the tumor's evolution at subclonal resolution. RESULTS: The most unique, previously unreported aspect of the tumor's evolution that we observed in this patient was the presence of "subclone incubators," defined as metastatic sites where substantial tumor evolution occurs before colonization of additional sites and organs by subclones that initially evolved at the incubator site. Overall, we identified four discrete waves of metastatic expansions, each of which resulted in a number of new, genetically similar metastasis sites that also enriched for particular organs (e.g., abdominal vs bone and brain). The lung played a critical role in facilitating metastatic spread in this patient: the lung was the first site of metastatic escape from the primary breast lesion, subclones at this site were likely the source of all four subsequent metastatic waves, and multiple sites in the lung acted as subclone incubators. Finally, functional annotation revealed that many known drivers or metastasis-promoting tumor mutations in this patient were shared by some, but not all metastatic sites, highlighting the need for more comprehensive surveys of a patient's metastases for effective clinical intervention. CONCLUSIONS: Our analysis revealed the presence of substantial tumor evolution at metastatic incubator sites in a patient, with potentially important clinical implications. Our study demonstrated that sampling of a large number of metastatic sites affords unprecedented detail for studying metastatic evolution.

Deep whole-genome sequencing of multiple proband tissues and parental blood reveals the complex genetic etiology of congenital diaphragmatic hernias.

The diaphragm is critical for respiration and separation of the thoracic and abdominal cavities, and defects in diaphragm development are the cause of congenital diaphragmatic hernias (CDH), a common and often lethal birth defect. The genetic etiology of CDH is complex. Single-nucleotide variants (SNVs), insertions/deletions (indels), and structural variants (SVs) in more than 150 genes have been associated with CDH, although few genes are recurrently mutated in multiple individuals and mutated genes are incompletely penetrant. This suggests that multiple genetic variants in combination, other not-yet-investigated classes of variants, and/or nongenetic factors contribute to CDH etiology. However, no studies have comprehensively investigated in affected individuals the contribution of all possible classes of variants throughout the genome to CDH etiology. In our study, we used a unique cohort of four individuals with isolated CDH with samples from blood, skin, and diaphragm connective tissue and parental blood and deep whole-genome sequencing to assess germline and somatic de novo and inherited SNVs, indels, and SVs. In each individual we found a different mutational landscape that included germline de novo and inherited SNVs and indels in multiple genes. We also found in two individuals a 343 bp deletion interrupting an annotated enhancer of the CDH-associated gene GATA4, and we hypothesize that this common SV (found in 1%-2% of the population) acts as a sensitizing allele for CDH. Overall, our comprehensive reconstruction of the genetic architecture of four CDH individuals demonstrates that the etiology of CDH is heterogeneous and multifactorial.

Low-Frequency (Gigahertz to Terahertz) Depolarized Raman Scattering Off n-Alkanes, Cycloalkanes, and Six-Membered Rings: A Physical Interpretation.

Molecular liquids have long been known to undergo various distinct intermolecular motions, from fast librations and cage-rattling oscillations to slow orientational and translational diffusion. However, their resultant gigahertz to terahertz spectra are far from simple, appearing as broad shapeless bands that span many orders of magnitude of frequency, making meaningful interpretation troublesome. Ad hoc spectral line shape fitting has become a notoriously fine art in the field; a unified approach to handling such spectra is long overdue. Here we apply ultrafast optical Kerr-effect (OKE) spectroscopy to study the intermolecular dynamics of room-temperature n-alkanes, cycloalkanes, and six-carbon rings, as well as liquid methane and propane. This work provides stress tests and converges upon an experimentally robust model across simple molecular series and range of temperatures, providing a blueprint for the interpretation of the dynamics of van der Waals liquids. This will enable the interpretation of low-frequency spectra of more complex liquids.

Genomic analyses implicate noncoding de novo variants in congenital heart disease.

A genetic etiology is identified for one-third of patients with congenital heart disease (CHD), with 8% of cases attributable to coding de novo variants (DNVs). To assess the contribution of noncoding DNVs to CHD, we compared genome sequences from 749 CHD probands and their parents with those from 1,611 unaffected trios. Neural network prediction of noncoding DNV transcriptional impact identified a burden of DNVs in individuals with CHD (n = 2,238 DNVs) compared to controls (n = 4,177; P = 8.7 × 10-4). Independent analyses of enhancers showed an excess of DNVs in associated genes (27 genes versus 3.7 expected, P = 1 × 10-5). We observed significant overlap between these transcription-based approaches (odds ratio (OR) = 2.5, 95% confidence interval (CI) 1.1-5.0, P = 5.4 × 10-3). CHD DNVs altered transcription levels in 5 of 31 enhancers assayed. Finally, we observed a DNV burden in RNA-binding-protein regulatory sites (OR = 1.13, 95% CI 1.1-1.2, P = 8.8 × 10-5). Our findings demonstrate an enrichment of potentially disruptive regulatory noncoding DNVs in a fraction of CHD at least as high as that observed for damaging coding DNVs.

Experimental observation of nanophase segregation in aqueous salt solutions around the predicted liquid-liquid transition in water.

The liquid-liquid transition in supercooled liquid water, predicted to occur around 220 K, is controversial due to the difficulty of studying it caused by competition from ice crystallization (the so-called "no man's land"). In aqueous solutions, it has been predicted to give rise to phase separation on a nanometer scale between a solute-rich high-density phase and a water-rich low-density phase. Here we report direct experimental evidence for the formation of a nanosegregated phase in eutectic aqueous solutions of LiCl and LiSCN where the presence of crystalline water can be experimentally excluded. Femtosecond infrared and Raman spectroscopies are used to determine the temperature-dependent structuring of water, the solvation of the SCN- anion, and the size of the phase segregated domains.

Polyamorphism Mirrors Polymorphism in the Liquid-Liquid Transition of a Molecular Liquid.

Liquid-liquid transitions between two amorphous phases in a single-component liquid have courted controversy. All known examples of liquid-liquid transitions in molecular liquids have been observed in the supercooled state, suggesting an intimate connection with vitrification and locally favored structures inhibiting crystallization. However, there is precious little information about the local molecular packing in supercooled liquids, meaning that the order parameter of the transition is still unknown. Here, we investigate the liquid-liquid transition in triphenyl phosphite and show that it is caused by the competition between liquid structures that mirror two crystal polymorphs. The liquid-liquid transition is found to be between a geometrically frustrated liquid and a dynamically frustrated glass. These results indicate a general link between polymorphism and polyamorphism and will lead to a much greater understanding of the physical basis of liquid-liquid transitions and allow the systematic discovery of other examples.

Pedigree-based estimation of human mobile element retrotransposition rates.

Germline mutation rates in humans have been estimated for a variety of mutation types, including single-nucleotide and large structural variants. Here, we directly measure the germline retrotransposition rate for the three active retrotransposon elements: L1, Alu, and SVA. We used three tools for calling mobile element insertions (MEIs) (MELT, RUFUS, and TranSurVeyor) on blood-derived whole-genome sequence (WGS) data from 599 CEPH individuals, comprising 33 three-generation pedigrees. We identified 26 de novo MEIs in 437 births. The retrotransposition rate estimates for Alu elements, one in 40 births, is roughly half the rate estimated using phylogenetic analyses, a difference in magnitude similar to that observed for single-nucleotide variants. The L1 retrotransposition rate is one in 63 births and is within range of previous estimates (1:20-1:200 births). The SVA retrotransposition rate, one in 63 births, is much higher than the previous estimate of one in 900 births. Our large, three-generation pedigrees allowed us to assess parent-of-origin effects and the timing of insertion events in either gametogenesis or early embryonic development. We find a statistically significant paternal bias in Alu retrotransposition. Our study represents the first in-depth analysis of the rate and dynamics of human retrotransposition from WGS data in three-generation human pedigrees.

Multi-platform discovery of haplotype-resolved structural variation in human genomes.

The incomplete identification of structural variants (SVs) from whole-genome sequencing data limits studies of human genetic diversity and disease association. Here, we apply a suite of long-read, short-read, strand-specific sequencing technologies, optical mapping, and variant discovery algorithms to comprehensively analyze three trios to define the full spectrum of human genetic variation in a haplotype-resolved manner. We identify 818,054 indel variants (<50 bp) and 27,622 SVs (≥50 bp) per genome. We also discover 156 inversions per genome and 58 of the inversions intersect with the critical regions of recurrent microdeletion and microduplication syndromes. Taken together, our SV callsets represent a three to sevenfold increase in SV detection compared to most standard high-throughput sequencing studies, including those from the 1000 Genomes Project. The methods and the dataset presented serve as a gold standard for the scientific community allowing us to make recommendations for maximizing structural variation sensitivity for future genome sequencing studies.

Phenotype of CM-AVM2 caused by variants in EPHB4: how much overlap with hereditary hemorrhagic telangiectasia (HHT)?

PURPOSE: EPHB4 variants were recently reported to cause capillary malformation-arteriovenous malformation 2 (CM-AVM2). CM-AVM2 mimics RASA1-related CM-AVM1 and hereditary hemorrhagic telangiectasia (HHT), as clinical features include capillary malformations (CMs), telangiectasia, and arteriovenous malformations (AVMs). Epistaxis, another clinical feature that overlaps with HHT, was reported in several cases. Based on the clinical overlap of CM-AVM2 and HHT, we hypothesized that patients considered clinically suspicious for HHT with no variant detected in an HHT gene (ENG, ACVRL1, or SMAD4) may have an EPHB4 variant. METHODS: Exome sequencing or a next-generation sequencing panel including EPHB4 was performed on individuals with previously negative molecular genetic testing for the HHT genes and/or RASA1. RESULTS: An EPHB4 variant was identified in ten unrelated cases. Seven cases had a pathogenic EPHB4 variant, including one with mosaicism. Three cases had an EPHB4 variant of uncertain significance. The majority had epistaxis (6/10 cases) and telangiectasia (8/10 cases), as well as CMs. Two of ten cases had a central nervous system AVM. CONCLUSIONS: Our results emphasize the importance of considering CM-AVM2 as part of the clinical differential for HHT and other vascular malformation syndromes. Yet, these cases highlight significant differences in the cutaneous presentations of CM-AVM2 versus HHT.

Rock Climber Self-Rescue Skills.

INTRODUCTION: Rock climbing involves some inherent danger, and rock climbers should be able to carry out basic rescue techniques for their own safety. This study seeks to assess such abilities by examining self-rescue skills in a cohort of rock climbers. METHODS: Climbers who participate in multipitch sport or traditional climbing styles were recruited via posters at a local climbing gym and on social media. Participants completed a survey assessing climbing history and confidence in their rescue skills and then were evaluated on 3 rescue scenarios in an indoor, standardized setting. Scenario pass rates were calculated and compared with rescue skill confidence on the survey. RESULTS: Twenty-five climbers participated in the study. Mean confidence in rescue skills varied from 4 to 4.5 (on a 7-point scale). The pass rates for the 3 scenarios were 28%, 68%, and 52%. Only 24% of climbers passed all 3 scenarios. Surveyed confidence in rescue skills and pass rate statistically correlated in only 1 scenario. CONCLUSIONS: Self-rescue skills were generally lacking in our study population. Climber confidence, experience, training, and climbing frequency did not appear to be associated with a higher level of rescue skills. Self-rescue skills should be emphasized in climbing instruction and courses to increase overall safety.