High accuracy genotyping directly from genomic DNA using a rolling circle amplification based assay

BACKGROUND: Rolling circle amplification of ligated probes is a simple and sensitive means for genotyping directly from genomic DNA. SNPs and mutations are interrogated with open circle probes (OCP) that can be circularized by DNA ligase when the probe matches the genotype. An amplified detection signal is generated by exponential rolling circle amplification (ERCA) of the circularized probe. The low cost and scalability of ligation/ERCA genotyping makes it ideally suited for automated, high throughput methods. RESULTS: A retrospective study using human genomic DNA samples of known genotype was performed for four different clinically relevant mutations: Factor V Leiden, Factor II prothrombin, and two hemochromatosis mutations, C282Y and H63D. Greater than 99% accuracy was obtained genotyping genomic DNA samples from hundreds of different individuals. The combined process of ligation/ERCA was performed in a single tube and produced fluorescent signal directly from genomic DNA in less than an hour. In each assay, the probes for both normal and mutant alleles were combined in a single reaction. Multiple ERCA primers combined with a quenched-peptide nucleic acid (Q-PNA) fluorescent detection system greatly accellerated the appearance of signal. Probes designed with hairpin structures reduced misamplification. Genotyping accuracy was identical from either purified genomic DNA or genomic DNA generated using whole genome amplification (WGA). Fluorescent signal output was measured in real time and as an end point. CONCLUSIONS: Combining the optimal elements for ligation/ERCA genotyping has resulted in a highly accurate single tube assay for genotyping directly from genomic DNA samples. Accuracy exceeded 99 % for four probe sets targeting clinically relevant mutations. No genotypes were called incorrectly using either genomic DNA or whole genome amplified sample

GDNF and Parkinson's Disease : Where Next? A Summary from a Recent Workshop

The concept of repairing the brain with growth factors has been pursued for many years in a variety of neurodegenerative diseases including primarily Parkinson's disease (PD) using glial cell line-derived neurotrophic factor (GDNF). This neurotrophic factor was discovered in 1993 and shown to have selective effects on promoting survival and regeneration of certain populations of neurons including the dopaminergic nigrostriatal pathway. These observations led to a series of clinical trials in PD patients including using infusions or gene delivery of GDNF or the related growth factor, neurturin (NRTN). Initial studies, some of which were open label, suggested that this approach could be of value in PD when the agent was injected into the putamen rather than the cerebral ventricles. In subsequent double-blind, placebo-controlled trials, the most recent reporting in 2019, treatment with GDNF did not achieve its primary end point. As a result, there has been uncertainty as to whether GDNF (and by extrapolation, related GDNF family neurotrophic factors) has merit in the future treatment of PD. To critically appraise the existing work and its future, a special workshop was held to discuss and debate this issue. This paper is a summary of that meeting with recommendations on whether there is a future for this therapeutic approach and also what any future PD trial involving GDNF and other GDNF family neurotrophic factors should consider in its design.Peer reviewe

A phenotype-structured model to reproduce the avascular growth of a tumor and its interaction with the surrounding environment

We here propose a one-dimensional spatially explicit phenotype-structured model to analyze selected aspects of avascular tumor progression. In particular, our approach distinguishes viable and necrotic cell fractions. The metabolically active part of the disease is, in turn, differentiated according to a continuous trait, that identifies cell variants with different degrees of motility and proliferation potential. A parabolic partial differential equation (PDE) then governs the spatio-temporal evolution of the phenotypic distribution of active cells within the host tissue. In this respect, active tumor agents are allowed to duplicate, move upon haptotactic and pressure stimuli, and eventually undergo necrosis. The mutual influence between the emerging malignancy and its environment (in terms of molecular landscape) is implemented by coupling the evolution law of the viable tumor mass with a parabolic PDE for oxygen kinetics and a differential equation that accounts for local consumption of extracellular matrix (ECM) elements. The resulting numerical realizations reproduce tumor growth and invasion in a number scenarios that differ for cell properties (i.e., individual migratory behavior, duplication, and mutation potential) and environmental conditions (i.e., level of tissue oxygenation and homogeneity in the initial matrix profile). In particular, our simulations show that, in all cases, more mobile cell variants occupy the front edge of the tumor, whereas more proliferative clones are selected at more internal regions. A necrotic core constantly occupies the bulk of the mass due to nutrient deprivation. This work may eventually suggest some biomedical strategies to partially reduce tumor aggressiveness, i.e., to enhance necrosis of malignant tissue and to promote the presence of more proliferative cell phenotypes over more invasive ones

Carcinoid tumor in a presacral teratoma associated with an anterior sacral meningocele: case report and review of the literature.

We report a case of a presacral teratoma containing a malignant carcinoid component associated with an anterior sacral meningocele that presented in a 35-year-old woman. The clinical, radiographic, and pathological features of these rare tumors, of presacral meningoceles, and of the hereditary presacral teratoma syndrome are discussed