Search

Across 20 vaccine breakthrough cases detected at our institution, all 20 (100%) infections were due to variants of concern (VOCs) and had a median Ct of 20.2 (IQR, 17.1–23.3). When compared with 5174 contemporaneous samples sequenced in our laboratory, VOCs were significantly enriched among breakthrough infections (P < .05).

A central problem in genomics is understanding the effect of individual DNA variants. Multiplexed Assays of Variant Effect (MAVEs) can help address this challenge by measuring all possible single nucleotide variant effects in a gene or regulatory sequence simultaneously. Here we describe MaveDB v2, which has become the database of record for MAVEs. MaveDB now contains a large fraction of published studies, comprising over two hundred datasets and three million variant effect measurements. We created tools and APIs to streamline data submission and access, transforming MaveDB into a hub for the analysis and dissemination of these impactful datasets.

Across 20 vaccine breakthrough cases detected at our institution, all 20 (100%) infections were due to variants of concern (VOC) and had a median Ct of 20.2 (IQR=17.1-23.3). When compared to 5174 contemporaneous samples sequenced in our laboratory, VOC were significantly enriched among breakthrough infections (p < .05).

Genetic data are rapidly becoming part of tumor classification and are integral to prognosis and predicting response to therapy. Current molecular tumor profiling relies heavily on tissue resection or biopsy. Tissue profiling has several disadvantages in tumors of the central nervous system, including the challenge associated with invasive biopsy, the heterogeneous nature of many malignancies where a small biopsy can underrepresent the mutational profile, and the frequent lack of obtaining a repeat biopsy, which limits routine monitoring to assess therapy response and/or tumor evolution. Circulating tumor, cell-free DNA (cfDNA), has been proposed as a liquid biopsy to address some limitations of tissue-based genetics. In cancer patients, a portion of cfDNA is tumor-derived and may contain somatic genetic alterations. In central nervous system (CNS) neoplasia, plasma tumor-derived cfDNA is very low or absent, likely due to the blood brain barrier. Interrogating cfDNA in cerebrospinal fluid (CSF) has several advantages. Compared to blood, CSF is paucicellular and therefore predominantly lacks non-tumor cfDNA; however, patients with CNS-limited tumors have significantly enriched tumor-derived cfDNA in CSF. In patients with metastatic CNS disease, mutations in CSF cfDNA are most concordant with the intracranial process. CSF cfDNA can also occasionally uncover additional genetic alterations absent in concurrent biopsy specimens, reflecting tumor heterogeneity. Although CSF is enriched for tumor-derived cfDNA, absolute quantities are low. Highly sensitive, targeted methods including next-generation sequencing and digital PCR are required to detect mutations in CSF cfDNA. Additional technical and bioinformatic approaches also facilitate enhanced ability to detect tumor mutations in CSF cfDNA.

Introduction Glioblastoma multiforme (GBM) is the most aggressive and most common primary brain tumor. Treatment of GBM and other high-grade gliomas consists of surgical resection followed by radiation and temozolomide (TMZ) chemotherapy. TMZ alkylates purine DNA bases, causing DNA damage and eventually cell death. Alkylation at O6-gauanine can be reversed by the DNA repair enzyme methylguanine methyltransferase (MGMT), inhibiting the desired cytotoxic effect. MGMT is epigenetically silenced by methylation of a CpG island in its promoter. Many high-grade gliomas are hypomethylated at the MGMT promoter. MGMT promoter methylation has emerged as a biomarker for response to alkylating chemotherapy. Multiple clinical trials demonstrate that the patients most likely to benefit from TMZ therapy have a hypermethylated MGMT promoter. MGMT promoter status is now incorporated into adjuvant therapy guidelines. Our institution receives approximately 70 to 80 high-grade glioma resections per year. The goal of this study is to validate a PCR-based MGMT promoter methylation assay and to compare results to those previously obtained at a well-known reference lab. Methods We extracted DNA from high-grade gliomas specimens (FFPE) or used commercially available methylated and unmethylated DNA controls. DNA was treated with bisulfite to convert unmethylated cytosine into uracil. Promoter methylation was detected with real-time quantitative PCR using a primer/probe set specific for methylated CpG sites within the MGMT promoter (meMGMT, published in Ida et al 2017) or to a housekeeping gene (COL2A1). Results Using control methylated DNA, we demonstrated that our assay consistently detects meMGMT and COL2A1 at DNA inputs greater than 5 ng. Both within- and between-run imprecision was minimal with little variation in crossing thresholds (Cts) for both COL2A1 (%CV at 50 ng input: 7.9% [within-run], 0.3% [between-run]) and MGMT (%CV at 50 ng input: 0.6% [within-run], 0.4% [between-run]). Delta Ct values were consistent across different levels of DNA input. Analysis of 15 cases from our archives (8 positive, 7 negative) demonstrates that our results (delta Ct values) correlate (R2 = 0.9) with the reference lab methylation score ( Conclusions Our PCR-based MGMT methylation assay has excellent analytical sensitivity and low imprecision. Preliminary analysis of patient samples shows that our results are concordant with historic reference lab results.

The cadherin–catenin complex mediates cell–cell adhesion at adherens junctions. Phosphorylation of E-cadherin in its β-catenin–binding domain promotes surface stability of E-cadherin and robust cell–cell adhesion., E-cadherin is highly phosphorylated within its β-catenin–binding region, and this phosphorylation increases its affinity for β-catenin in vitro. However, the identification of key serines responsible for most cadherin phosphorylation and the adhesive consequences of modification at such serines have remained unknown. In this study, we show that as few as three serines in the β-catenin–binding domain of E-cadherin are responsible for most radioactive phosphate incorporation. These serines are required for binding to β-catenin and the mutual stability of both E-cadherin and β-catenin. Cells expressing a phosphodeficient (3S>A) E-cadherin exhibit minimal cell–cell adhesion due to enhanced endocytosis and degradation through a lysosomal compartment. Conversely, negative charge substitution at these serines (3S>D) antagonizes cadherin endocytosis and restores wild-type levels of adhesion. The cadherin kinase is membrane proximal and modifies the cadherin before it reaches the cell surface. Together these data suggest that E-cadherin phosphorylation is largely constitutive and integral to cadherin–catenin complex formation, surface stability, and function.

The cadherin/catenin complex organizes to form a structural Velcro that joins the cytoskeletal networks of adjacent cells. Functional loss of this complex arrests the development of normal tissue organization, and years of research have gone into teasing out how the physical structure of adhesions conveys information to the cell interior. Evidence that most cadherin-binding partners also localize to the nucleus to regulate transcription supports the view that cadherins serve as simple stoichiometric inhibitors of nuclear signals. However, it is also clear that cadherin-based adhesion initiates a variety of molecular events that can ultimately impact nuclear signaling. This chapter discusses these two modes of cadherin signaling in the context of tissue growth and differentiation.

Agarose-gel electrophoresis has been used for more than thirty years to characterize the linking-number (Lk) distribution of closed-circular DNA molecules. Although the physical basis of this technique remains poorly understood, the gel-electrophoretic behavior of covalently closed DNAs has been used to determine the local unwinding of DNA by proteins and small-molecule ligands, characterize supercoiling-dependent conformational transitions in duplex DNA, and to measure helical-repeat changes due to shifts in temperature and ionic strength. Those results have been analyzed by assuming that the absolute mobility of a particular topoisomer is mainly a function of the integral number of superhelical turns, and thus a slowly varying function of plasmid molecular weight. In examining the mobilities of Lk topoisomers for a series of plasmids that differ incrementally in size over more than one helical turn, we found that the size-dependent agarose-gel mobility of individual topoisomers with identical values of Lk (but different values of the excess linking number, DeltaLk) vary dramatically over a duplex turn. Our results suggest that a simple semi-empirical relationship holds between the electrophoretic mobility of linking-number topoisomers and their average writhe in solution.

The lack of a rigorous analytical theory for DNA looping has caused many DNA-loop-mediated phenomena to be interpreted using theories describing the related process of DNA cyclization. However, distinctions in the mechanics of DNA looping versus cyclization can have profound quantitative effects on the thermodynamics of loop closure. We have extended a statistical mechanical theory recently developed for DNA cyclization to model DNA looping, taking into account protein flexibility. Notwithstanding the underlying theoretical similarity, we find that the topological constraint of loop closure leads to the coexistence of multiple classes of loops mediated by the same protein structure. These loop topologies are characterized by dramatic differences in twist and writhe; because of the strong coupling of twist and writhe within a loop, DNA looping can exhibit a complex overall helical dependence in terms of amplitude, phase, and deviations from uniform helical periodicity. Moreover, the DNA-length dependence of optimal looping efficiency depends on protein elasticity, protein geometry, and the presence of intrinsic DNA bends. We derive a rigorous theory of loop formation that connects global mechanical and geometric properties of both DNA and protein and demonstrates the importance of protein flexibility in loop-mediated protein-DNA interactions.

Interactions of E. coli lac repressor (LacR) with a pair of operator sites on the same DNA molecule can lead to the formation of looped nucleoprotein complexes both in vitro and in vivo. As a major paradigm for loop-mediated gene regulation, parameters such as operator affinity and spacing, repressor concentration, and DNA bending induced by specific or non-specific DNA-binding proteins (e.g., HU), have been examined extensively. However, a complete and rigorous model that integrates all of these aspects in a systematic and quantitative treatment of experimental data has not been available. Applying our recent statistical-mechanical theory for DNA looping, we calculated repression as a function of operator spacing (58-156 bp) from first principles and obtained excellent agreement with independent sets of in-vivo data. The results suggest that a linear extended, as opposed to a closed v-shaped, LacR conformation is the dominant form of the tetramer in vivo. Moreover, loop-mediated repression in wild-type E. coli strains is facilitated by decreased DNA rigidity and high levels of flexibility in the LacR tetramer. In contrast, repression data for strains lacking HU gave a near-normal value of the DNA persistence length. These findings underscore the importance of both protein conformation and elasticity in the formation of small DNA loops widely observed in vivo, and demonstrate the utility of quantitatively analyzing gene regulation based on the mechanics of nucleoprotein complexes.