scholarly journals Ultra–sensitive droplet digital PCR for detecting a low–prevalence somatic GNAQ mutation in Sturge–Weber syndrome

2016 ◽  
Vol 6 (1) ◽  
Author(s):  
Yuri Uchiyama ◽  
Mitsuko Nakashima ◽  
Satoshi Watanabe ◽  
Masakazu Miyajima ◽  
Masataka Taguri ◽  
...  
Keyword(s):  
Detection Limit ◽  
Nucleic Acid ◽  
Somatic Mutation ◽  
Digital Pcr ◽  
Droplet Digital Pcr ◽  
Blood Leukocytes ◽  
Target Nucleic Acid ◽  
Weber Syndrome ◽  
Sturge Weber Syndrome ◽  
Blood Leukocyte

Abstract Droplet digital PCR (ddPCR), a method for measuring target nucleic acid sequence quantity, is useful for determining somatic mutation rates using TaqMan probes. In this study, the detection limit of copy numbers of test DNA by ddPCR was determined based on Poisson distribution. Peptide nucleic acid (PNA), which strongly hybridises to target lesions, can inhibit target amplification by PCR. Therefore, by combination of PCR with PNA and ddPCR (PNA–ddPCR), the detection limit could be lowered. We reanalysed a somatic GNAQ mutation (c.548G > A) in patients with Sturge–Weber syndrome (SWS) using ddPCR and PNA–ddPCR. Importantly, among three patients previously found to be mutation negative by next–generation sequencing, two patients had the GNAQ mutation with a mutant allele frequency of less than 1%. Furthermore, we were able to find the same mutation in blood leukocyte or saliva DNA derived from four out of 40 SWS patients. Vascular anomalies and blood leukocytes originate from endothelial cells and haemangioblasts, respectively, which are both of mesodermal origin. Therefore, blood leukocytes may harbour the GNAQ mutation, depending on the time when the somatic mutation is acquired. These data suggest the possibility of diagnosis using blood DNA in some patients with SWS.

scholarly journals Somatic GNAQ mutation in the forme fruste of Sturge-Weber syndrome

2018 ◽  
Vol 4 (3) ◽  
pp. e236 ◽  
Author(s):  
Michael S. Hildebrand ◽  
A. Simon Harvey ◽  
Stephen Malone ◽  
John A. Damiano ◽  
Hongdo Do ◽  
...  
Keyword(s):  
Brain Tissue ◽  
Molecular Mechanism ◽  
Digital Pcr ◽  
Droplet Digital Pcr ◽  
Cutaneous Manifestations ◽  
Weber Syndrome ◽  
Sturge Weber Syndrome ◽  
Low Levels ◽  
The Brain ◽  
Molecular Evaluation

ObjectiveTo determine whether the GNAQ R183Q mutation is present in the forme fruste cases of Sturge-Weber syndrome (SWS) to establish a definitive molecular diagnosis.MethodsWe used sensitive droplet digital PCR (ddPCR) to detect and quantify the GNAQ mutation in tissues from epilepsy surgery in 4 patients with leptomeningeal angiomatosis; none had ocular or cutaneous manifestations.ResultsLow levels of the GNAQ mutation were detected in the brain tissue of all 4 cases—ranging from 0.42% to 7.1% frequency—but not in blood-derived DNA. Molecular evaluation confirmed the diagnosis in 1 case in which the radiologic and pathologic data were equivocal.ConclusionsWe detected the mutation at low levels, consistent with mosaicism in the brain or skin (1.0%–18.1%) of classic cases. Our data confirm that the forme fruste is part of the spectrum of SWS, with the same molecular mechanism as the classic disease and that ddPCR is helpful where conventional diagnosis is uncertain.

scholarly journals Erratum: Corrigendum: Ultra–sensitive droplet digital PCR for detecting a low–prevalence somatic GNAQ mutation in Sturge–Weber syndrome

2017 ◽  
Vol 7 (1) ◽  
Author(s):  
Yuri Uchiyama ◽  
Mitsuko Nakashima ◽  
Satoshi Watanabe ◽  
Masakazu Miyajima ◽  
Masataka Taguri ◽  
...  
Keyword(s):  
Digital Pcr ◽  
Droplet Digital Pcr ◽  
Weber Syndrome ◽  
Sturge Weber Syndrome ◽  
Low Prevalence

scholarly journals Sturge–Weber Syndrome and Port-Wine Stains Caused by Somatic Mutation in GNAQ

2013 ◽  
Vol 368 (21) ◽  
pp. 1971-1979 ◽  
Author(s):  
Matthew D. Shirley ◽  
Hao Tang ◽  
Carol J. Gallione ◽  
Joseph D. Baugher ◽  
Laurence P. Frelin ◽  
...  
Keyword(s):  
Somatic Mutation ◽  
Port Wine ◽  
Port Wine Stains ◽  
Weber Syndrome ◽  
Sturge Weber Syndrome

scholarly journals Development of SARS-CoV-2 packaged RNA reference material for nucleic acid testing

2021 ◽  
Author(s):  
Sang-Soo Lee ◽  
Seil Kim ◽  
Hee Min Yoo ◽  
Da-Hye Lee ◽  
Young-Kyung Bae
Keyword(s):  
Nucleic Acid ◽  
Copy Number ◽  
Target Gene ◽  
Target Genes ◽  
Digital Pcr ◽  
Number Concentration ◽  
Droplet Digital Pcr ◽  
Molecular Testing ◽  
Testing Laboratories ◽  
Viral Target

AbstractNucleic acid tests to detect the SARS-CoV-2 virus have been performed worldwide since the beginning of the COVID-19 pandemic. For the quality assessment of testing laboratories and the performance evaluation of molecular diagnosis products, reference materials (RMs) are required. In this work, we report the production of a lentiviral SARS-CoV-2 RM containing approximately 12 kilobases of its genome including common diagnostics targets such as RdRp, N, E, and S genes. The RM was measured with multiple assays using two different digital PCR platforms. To measure the homogeneity and stability of the lentiviral SARS-CoV-2 RM, reverse transcription droplet digital PCR (RT-ddPCR) was used with in-house duplex assays. The copy number concentration of each target gene in the extracted RNA solution was then converted to that of the RM solution. Their copy number values are measured to be from 1.5 × 105 to 2.0 × 105 copies/mL. The RM has a between-bottle homogeneity of 4.80–8.23% and is stable at 4 °C for 1 week and at −70 °C for 6 months. The lentiviral SARS-CoV-2 RM closely mimics real samples that undergo identical pre-analytical processes for SARS-CoV-2 molecular testing. By offering accurate reference values for the absolute copy number of viral target genes, the developed RM can be used to improve the reliability of SARS-CoV-2 molecular testing.

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