Inverted triplications formed by iterative template switches generate structural variant diversity at genomic disorder loci.

The duplication-triplication/inverted-duplication (DUP-TRP/INV-DUP) structure is a complex genomic rearrangement (CGR). Although it has been identified as an important pathogenic DNA mutation signature in genomic disorders and cancer genomes, its architecture remains unresolved. Here, we studied the genomic architecture of DUP-TRP/INV-DUP by investigating the DNA of 24 patients identified by array comparative genomic hybridization (aCGH) on whom we found evidence for the existence of 4 out of 4 predicted structural variant (SV) haplotypes. Using a combination of short-read genome sequencing (GS), long-read GS, optical genome mapping, and single-cell DNA template strand sequencing (strand-seq), the haplotype structure was resolved in 18 samples. The point of template switching in 4 samples was shown to be a segment of ∼2.2-5.5 kb of 100% nucleotide similarity within inverted repeat pairs. These data provide experimental evidence that inverted low-copy repeats act as recombinant substrates. This type of CGR can result in multiple conformers generating diverse SV haplotypes in susceptible dosage-sensitive loci.

Break-induced replication underlies formation of inverted triplications and generates unexpected diversity in haplotype structures.

Background: The duplication-triplication/inverted-duplication (DUP-TRP/INV-DUP) structure is a type of complex genomic rearrangement (CGR) hypothesized to result from replicative repair of DNA due to replication fork collapse. It is often mediated by a pair of inverted low-copy repeats (LCR) followed by iterative template switches resulting in at least two breakpoint junctions in cis . Although it has been identified as an important mutation signature of pathogenicity for genomic disorders and cancer genomes, its architecture remains unresolved and is predicted to display at least four structural variation (SV) haplotypes. Results: Here we studied the genomic architecture of DUP-TRP/INV-DUP by investigating the genomic DNA of 24 patients with neurodevelopmental disorders identified by array comparative genomic hybridization (aCGH) on whom we found evidence for the existence of 4 out of 4 predicted SV haplotypes. Using a combination of short-read genome sequencing (GS), long- read GS, optical genome mapping and StrandSeq the haplotype structure was resolved in 18 samples. This approach refined the point of template switching between inverted LCRs in 4 samples revealing a DNA segment of ∼2.2-5.5 kb of 100% nucleotide similarity. A prediction model was developed to infer the LCR used to mediate the non-allelic homology repair. Conclusions: These data provide experimental evidence supporting the hypothesis that inverted LCRs act as a recombinant substrate in replication-based repair mechanisms. Such inverted repeats are particularly relevant for formation of copy-number associated inversions, including the DUP-TRP/INV-DUP structures. Moreover, this type of CGR can result in multiple conformers which contributes to generate diverse SV haplotypes in susceptible loci .

Payment Methods and Demographics Influence Patterns of Dental Service Utilization.

OBJECTIVE: To describe the patterns of specific dental service utilization among the various sociodemographic groups in North Carolina served by the East Carolina University School of Dental Medicine (ECU SoDM). DESIGN: This was a descriptive study utilizing self-reported patients' sociodemographic information, payment method history, and CDT codes of procedures performed. Deidentified clinical data recorded for 26 710 patients and 534 983 procedures from 2011 to 2020 were extracted from a centralized axiUm database. Data were analyzed using IBM SPSS Statistics, version 25.0. Cross-tabulations between dental service utilizations, patients' demographics, and payment method were performed using chi-square analysis. SETTING: Nine dental clinic sites across the state of North Carolina. PARTICIPANTS: In total, 26 710 adults 23 years to older than 65 years were included in the sample for this study. MAIN OUTCOME MEASURES: In total, 534 983 procedure codes completed for the eligible patients were cross-tabulated with payment method. RESULTS: Payment method was significantly related to individual characteristics including location of service, age, race, ethnicity, and untreated decay ( P < .001). Payment method is associated with the dental service type utilized by an individual ( P < .001). Patients who received Medicaid benefits were more likely to receive restorative procedures, removable prosthetics, or oral surgery. Despite NC Medicaid covering preventive procedures, patients who received Medicaid benefits showed lower utilization of preventive procedures than expected. Privately insured or self-paying individuals demonstrated a greater variety of service option utilization, as well as more frequent usage of more specialized procedure options such as endodontics, periodontics, fixed prosthodontics, and implants. CONCLUSIONS: Payment method was found to be related to patients' demographics and type of dental service utilized. Adults older than 65 years demonstrated a higher proportion of self-payment for dental care, indicating a lack of payment options for this population. In the interest of providing care for underserved populations in North Carolina, policy makers should consider expanding dental coverage for adults older than 65 years.

Identifying synergistic high-order 3D chromatin conformations from genome-scale nanopore concatemer sequencing.

High-order three-dimensional (3D) interactions between more than two genomic loci are common in human chromatin, but their role in gene regulation is unclear. Previous high-order 3D chromatin assays either measure distant interactions across the genome or proximal interactions at selected targets. To address this gap, we developed Pore-C, which combines chromatin conformation capture with nanopore sequencing of concatemers to profile proximal high-order chromatin contacts at the genome scale. We also developed the statistical method Chromunity to identify sets of genomic loci with frequencies of high-order contacts significantly higher than background ('synergies'). Applying these methods to human cell lines, we found that synergies were enriched in enhancers and promoters in active chromatin and in highly transcribed and lineage-defining genes. In prostate cancer cells, these included binding sites of androgen-driven transcription factors and the promoters of androgen-regulated genes. Concatemers of high-order contacts in highly expressed genes were demethylated relative to pairwise contacts at the same loci. Synergies in breast cancer cells were associated with tyfonas, a class of complex DNA amplicons. These results rigorously link genome-wide high-order 3D interactions to lineage-defining transcriptional programs and establish Pore-C and Chromunity as scalable approaches to assess high-order genome structure.

Impact of HCV core gene quasispecies on hepatocellular carcinoma risk among HALT-C trial patients.

Mutations at positions 70 and/or 91 in the core protein of genotype-1b, hepatitis C virus (HCV) are associated with hepatocellular carcinoma (HCC) risk in Asian patients. To evaluate this in a US population, the relationship between the percentage of 70 and/or 91 mutant HCV quasispecies in baseline serum samples of chronic HCV patients from the HALT-C trial and the incidence of HCC was determined by deep sequencing. Quasispecies percentage cut-points, ≥42% of non-arginine at 70 (non-R(70)) or ≥98.5% of non-leucine at 91 (non-L(91)) had optimal sensitivity at discerning higher or lower HCC risk. In baseline samples, 88.5% of chronic HCV patients who later developed HCC and 68.8% of matched HCC-free control patients had ≥42% non-R(70) quasispecies (P = 0.06). Furthermore, 30.8% of patients who developed HCC and 54.7% of matched HCC-free patients had quasispecies with ≥98.5% non-L(91) (P = 0.06). By Kaplan-Meier analysis, HCC incidence was higher, but not statistically significant, among patients with quasispecies ≥42% non-R(70) (P = 0.08), while HCC incidence was significantly reduced among patients with quasispecies ≥98.5% non-L(91) (P = 0.01). In a Cox regression model, non-R(70) ≥42% was associated with increased HCC risk. This study of US patients indicates the potential utility of HCV quasispecies analysis as a non-invasive biomarker of HCC risk.

Characterizing and Overriding the Structural Mechanism of the Quizartinib-Resistant FLT3 "Gatekeeper" F691L Mutation with PLX3397.

UNLABELLED: Tyrosine kinase domain mutations are a common cause of acquired clinical resistance to tyrosine kinase inhibitors (TKI) used to treat cancer, including the FLT3 inhibitor quizartinib. Mutation of kinase "gatekeeper" residues, which control access to an allosteric pocket adjacent to the ATP-binding site, has been frequently implicated in TKI resistance. The molecular underpinnings of gatekeeper mutation-mediated resistance are incompletely understood. We report the first cocrystal structure of FLT3 with the TKI quizartinib, which demonstrates that quizartinib binding relies on essential edge-to-face aromatic interactions with the gatekeeper F691 residue, and F830 within the highly conserved Asp-Phe-Gly motif in the activation loop. This reliance makes quizartinib critically vulnerable to gatekeeper and activation loop substitutions while minimizing the impact of mutations elsewhere. Moreover, we identify PLX3397, a novel FLT3 inhibitor that retains activity against the F691L mutant due to a binding mode that depends less vitally on specific interactions with the gatekeeper position. SIGNIFICANCE: We report the first cocrystal structure of FLT3 with a kinase inhibitor, elucidating the structural mechanism of resistance due to the gatekeeper F691L mutation. PLX3397 is a novel FLT3 inhibitor with in vitro activity against this mutation but is vulnerable to kinase domain mutations in the FLT3 activation loop.

Multifocal pulmonary aspergillomas: case series and review.

Multifocal lung parenchymal cavities containing multiple aspergillomas are not well-recognized features of chronic pulmonary aspergillosis (CPA). We identified five patients with multiple cavities containing fungal balls from our accumulated cohort of ∼300 patients with CPA. Corticosteroid exposure and radiological and serological characteristics were assessed. The patients, aged 19-55 years, developed 3-11 cavities (or nodules in one case) with thin walls, usually within the lung parenchyma, with very limited pleural involvement. Four had asthma (severe in three) and one had cystic fibrosis; three had allergic bronchopulmonary aspergillosis. All patients had received corticosteroids orally or by inhalation. Four patients had elevated Aspergillus IgG antibodies; one had elevated Aspergillus-specific IgE. Three patients developed azole resistance on antifungal therapy, after benefit, one of whom underwent a successful bilateral lung transplant, later complicated by a fatal mycotic cerebral aneurysm. Multiple aspergillomas is a new distinct manifestation of CPA. The lack of inflammatory response and the distribution of the cavities in the lungs are remarkable. Aspergillus nodules could evolve into cavities with aspergillomas. The management and development of azole resistance in these patients is problematic.