A Simple, Universal, and Cost-Efficient Digital PCR Method for the Targeted Analysis of Copy Number Variations

Cassinari, Kévin; Quenez, Olivier; Beaussire, Ludivine; Meur, Nathalie Le; Castelain, Mathieu; Goldenberg, Alice; Guerrot, Anne‐Marie; Bréhin, Anne-Claire; Deleuze, Jean‐François; Boland, Anne; Rovelet‐Lecrux, Anne; Campion, Dominique; Saugier-Veber, Pascale; Gruchy, Nicolas; Frébourg, Thierry; Nicolas, Gaël; Sarafan-Vasseur, Nasrin; Chambon, Pascal

doi:10.1373/clinchem.2019.304246

Cited by 12 publications

(13 citation statements)

References 22 publications

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“…It is indeed considered as a fast, robust, reliable, cost-effective method that avoids the design of new hydrolysis probes for each new assay. 34 These technical advantages make RT-ddPCR easier to use routinely. In addition, we provide here the results of Pi uptake in PBMCs of one carrier with the aim to assess the protein function level.…”

Section: Discussionmentioning

confidence: 99%

Haploinsufficiency of the Primary Familial Brain Calcification Gene SLC20A2 Mediated by Disruption of a Regulatory Element

et al. 2020

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Primary familial brain calcification (PFBC) is a rare cerebral microvascular calcifying disorder with diverse neuropsychiatric expression. Five genes were reported as PFBC causative when carrying pathogenic variants. Haploinsufficiency of SLC20A2, which encodes an inorganic phosphate importer, is a major cause of autosomal-dominant PFBC. However, PFBC remains genetically unexplained in a proportion of patients, suggesting the existence of additional genes or cryptic mutations. We analyzed exome sequencing data of 71 unrelated, genetically unexplained PFBC patients with the aim to detect copy number variations that may disrupt the expression of core PFBC-causing genes. Methods: After the identification of a deletion upstream of SLC20A2, we assessed its consequences on gene function by reverse transcriptase droplet digital polymerase chain reaction (RT-ddPCR), an ex vivo inorganic

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Section: Discussionmentioning

confidence: 99%

Haploinsufficiency of the Primary Familial Brain Calcification Gene SLC20A2 Mediated by Disruption of a Regulatory Element

et al. 2020

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“…Following step 1, we implemented our workflow in our routine procedures. Form additional 1056 WES (supplementary table 1), we performed targeted confirmations following the detection of candidate CNVs by CANOES using QMPSF or ddPCR (16). We focused our confirmations on a list of 350 genes that belong to the so-called Aβ network (17), as all the samples used at this step were sequenced in the context of Alzheimer disease research.…”

Section: Assessment Of Cnv Calls From Whole Exome Sequencing Data: Stmentioning

confidence: 99%

Detection of copy-number variations from NGS data using read depth information: a diagnostic performance evaluation

Quenez

Cassinari²,

Coutant³

et al. 2020

Eur J Hum Genet

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The detection of Copy Number Variations (CNVs) from NGS data is under-exploited as chip-based or targeted techniques are still commonly used. We assessed the performances of a workflow centered on CANOES, a bioinformatics tool based on read depth information. We applied our workflow to gene panel (GP) and Whole Exome Sequencing (WES) data, and compared CNV calls to Quantitative Multiplex PCR of Short Fluorescent fragments (QMSPF) or array Comparative Genomic Hybridization (aCGH) results. From GP data of 3,776 samples, we reached an overall Positive Predictive Value (PPV) of 87.8%. This dataset included a complete comprehensive QMPSF comparison of 4 genes (60 exons) on which we obtained 100% sensitivity and specificity. From WES data, we first compared 137 samples to aCGH and filtered comparable events (exonic CNVs encompassing enough aCGH probes) and obtained an 87.25% sensitivity. The overall PPV was 86.4% following the targeted confirmation of candidate CNVs from 1,056 additional WES. In addition, our CANOES-centered workflow on WES data allowed the detection of CNVs of any size that were missed by aCGH. Overall, switching to a NGS-only approach should be costeffective as it allows a reduction in overall costs together with likely stable diagnostic yields. Our bioinformatics pipeline is available at : https://gitlab.bioinfo-diag.fr/nc4gpm/canoes-centeredworkflow.

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“…According to recently published methods of dPCR using universal LNA-hydrolysis probes from the 96 Universal Probe Library® (UPL, Sigma-Aldrich®, St. Louis, Missouri, USA), three dPCR assays were performed for each tumor sample [16]. These assays used a duplex PCR: one PCR amplicon within the EGFR gene and one reference PCR amplicon located in the hydroxymethylbilane synthase (HMBS) gene, a housekeeping gene located in 11q23.…”

Section: Development Of Dpcr Assaymentioning

confidence: 99%

“…The development of a specific molecular method allowing the simultaneous detection of EGFR alterations may be of interest in glioblastoma. Targeted copy number variation (CNV) detection by digital PCR (dPCR) using locked nucleic acid (LNA)-based hydrolysis probes has recently been shown to be efficient in genetic diseases [16]. LNAhydrolysis probes are very short nucleotides, and repeated sequences across the human genome may be incorporated in a dPCR amplicon.…”

Section: Introductionmentioning

confidence: 99%

Simultaneous detection of EGFR amplification and EGFRvIII variant using digital PCR-based method in glioblastoma

Fontanilles

Marguet

Ruminy

et al. 2020

acta neuropathol commun

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Epidermal growth factor receptor (EGFR) amplification and EGFR variant III (EGFRvIII, deletion of exons 2-7) are of clinical interest for glioblastoma. The aim was to develop a digital PCR (dPCR)-based method using locked nucleic acid (LNA)-based hydrolysis probes, allowing the simultaneous detection of the EGFR amplification and EGFRvIII variant. Sixty-two patients were included. An exploratory cohort (n = 19) was used to develop the dPCR assay using three selected amplicons within the EGFR gene, targeting intron 1 (EGFR1), junction of exon 3 and intron 3 (EGFR2) and intron 22 (EGFR3). The copy number of EGFR was estimated by the relative quantification of EGFR1, EGFR2 and EGFR3 amplicon droplets compared to the droplets of a reference gene. EGFRvIII was identified by comparing the copy number of the EGFR2 amplicon to either the EGFR1 or EGFR3 amplicon. dPCR results were compared to fluorescence in situ hybridization (FISH) and next-generation sequencing for amplification; and to RT-PCR-based method for EGFRvIII. The dPCR assay was then tested in a validation cohort (n = 43). A total of 8/19 EGFR-amplified and 5/19 EGFRvIII-positive tumors were identified in the exploratory cohort. Compared to FISH, the EGFR3 dPCR assay detected all EGFR-amplified tumors (8/8, 100%) and had the highest concordance with the copy number estimation by NGS. The concordance between RT-PCR and dPCR was also 100% for detecting EGFRvIII using an absolute difference of 10.8 for the copy number between EGFR2 and EGFR3 probes. In the validation cohort, the sensitivity and specificity of dPCR using EGFR3 probes were 100% for the EGFR amplification detection compared to FISH (19/19). EGFRvIII was detected by dPCR in 8 EGFR-amplified patients and confirmed by RT-PCR. Compared to FISH, the EGFR2/EGFR3 dPCR assay was estimated with a one-half cost value. These results highlight that dPCR allowed the simultaneous detection of EGFR amplification and EGFRvIII for glioblastoma.

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A Simple, Universal, and Cost-Efficient Digital PCR Method for the Targeted Analysis of Copy Number Variations

Cited by 12 publications

References 22 publications

Haploinsufficiency of the Primary Familial Brain Calcification Gene SLC20A2 Mediated by Disruption of a Regulatory Element

Haploinsufficiency of the Primary Familial Brain Calcification Gene SLC20A2 Mediated by Disruption of a Regulatory Element

Detection of copy-number variations from NGS data using read depth information: a diagnostic performance evaluation

Simultaneous detection of EGFR amplification and EGFRvIII variant using digital PCR-based method in glioblastoma

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