scholarly journals Development and validation of a 4-color multiplexing spinal muscular atrophy (SMA) genotyping assay on a novel integrated digital PCR instrument

2020 ◽  
Vol 10 (1) ◽  
Author(s):  
Lingxia Jiang ◽  
Robert Lin ◽  
Steve Gallagher ◽  
Andrew Zayac ◽  
Matthew E. R. Butchbach ◽  
...  
Keyword(s):  
Spinal Muscular Atrophy ◽  
Copy Number ◽  
Muscular Atrophy ◽  
Digital Pcr ◽  
Copy Number Variations ◽  
Higher Order ◽  
Clinical Samples ◽  
Internal Reference ◽  
Genotyping Assay ◽  
Copy Numbers

AbstractDigital PCR (dPCR) technology has been proven to be highly sensitive and accurate in detecting copy number variations (CNV). However, a higher-order multiplexing dPCR assay for measuring SMN1 and SMN2 copy numbers in spinal muscular atrophy (SMA) samples has not been reported. Described here is a rapid multiplex SMA dPCR genotyping assay run on a fully integrated dPCR instrument with five optical channels. The hydrolysis probe-based multiplex dPCR assay quantifies SMN1, SMN2, and the total SMN (SMN1 + SMN2) while using RPPH1 gene as an internal reference control. The quadruplex assay was evaluated with characterized control DNA samples and validated with 15 blinded clinical samples from a previously published study. SMN1 and SMN2 copy numbers were completely concordant with previous results for both the control and blinded samples. The dPCR-based SMA copy number determination was accomplished in 90 min with a walk-away workflow identical to real-time quantitative PCR (qPCR). In summary, presented here is a simple higher-order multiplexing solution on a novel digital PCR platform to meet the growing demand for SMA genotyping and prognostics.

scholarly journals Multiplex Droplet Digital PCR Method Applicable to Newborn Screening, Carrier Status, and Assessment of Spinal Muscular Atrophy

2018 ◽  
Vol 64 (12) ◽  
pp. 1753-1761 ◽  
Author(s):  
Noemi Vidal-Folch ◽  
Dimitar Gavrilov ◽  
Kimiyo Raymond ◽  
Piero Rinaldo ◽  
Silvia Tortorelli ◽  
...  
Keyword(s):  
Spinal Muscular Atrophy ◽  
Newborn Screening ◽  
Copy Number ◽  
Neuronal Degeneration ◽  
Muscular Atrophy ◽  
Simultaneous Detection ◽  
Digital Pcr ◽  
Droplet Digital Pcr ◽  
Carrier Status ◽  
Survival Motor Neuron Gene

Abstract BACKGROUND Spinal muscular atrophy (SMA) is a progressive neuromuscular disorder with neuronal degeneration leading to muscular atrophy and respiratory failure. SMA is frequently caused by homozygous deletions that include exon 7 of the survival motor neuron gene SMN1, and its clinical course is influenced by the copy number of a nearby 5q SMN1 paralog, SMN2. Multiple ligation probe amplification (MLPA) and real-time quantitative PCR (qPCR) can detect SMN1 deletions. Yet, qPCR needs normalization or standard curves, and MLPA demands DNA concentrations above those obtainable from dried blood spots (DBSs). We developed a multiplex, droplet digital PCR (ddPCR) method for the simultaneous detection of SMN1 deletions and SMN2 copy number variation in DBS and other tissues. An SMN1 Sanger sequencing process for DBS was also developed. METHODS SMN1, SMN2, and RPP30 concentrations were simultaneously measured with a Bio-Rad AutoDG and QX200 ddPCR system. A total of 1530 DBSs and 12 SMA patients were tested. RESULTS Population studies confirmed 1 to 5 SMN1 exon 7 copies detected in unaffected specimens, whereas patients with SMA revealed 0 SMN1 copies. Intraassay and interassay imprecisions were <7.1% CV for individuals with ≥1 SMN1 copies. Testing 12 SMA-positive samples resulted in 100% sensitivity and specificity. CONCLUSIONS This ddPCR method is sensitive, specific, and applicable to newborn screening and carrier status determination for SMA. It can also be incorporated with a parallel ddPCR T-cell excision circles assay for severe combined immunodeficiencies.

scholarly journals SMN1 and SMN2 copy numbers in cell lines derived from patients with spinal muscular atrophy as measured by array digital PCR

2015 ◽  
Vol 3 (4) ◽  
pp. 248-257 ◽  
Author(s):  
Deborah L. Stabley ◽  
Ashlee W. Harris ◽  
Jennifer Holbrook ◽  
Nicholas J. Chubbs ◽  
Kevin W. Lozo ◽  
...  
Keyword(s):  
Spinal Muscular Atrophy ◽  
Cell Lines ◽  
Muscular Atrophy ◽  
Digital Pcr ◽  
Copy Numbers

SMN2 copy number predicts acute or chronic spinal muscular atrophy but does not account for intrafamilial variability in siblings

2005 ◽  
Vol 253 (1) ◽  
pp. 21-25 ◽  
Author(s):  
I. Cuscó ◽  
M. J. Barceló ◽  
R. Rojas–García ◽  
I. Illa ◽  
J. Gámez ◽  
...  
Keyword(s):  
Spinal Muscular Atrophy ◽  
Copy Number ◽  
Muscular Atrophy ◽  
Intrafamilial Variability ◽  
Chronic Spinal Muscular Atrophy

Abstract P613: Absolute Gene Quantification Of Intrarenal Renin-angiotensin System Components Using Droplet Digital PCR Analysis

Hypertension ◽  
2016 ◽  
Vol 68 (suppl_1) ◽  
Author(s):  
Ryousuke Satou ◽  
Akemi Katsurada ◽  
Kayoko Miyata ◽  
Andrei Derbenev ◽  
Andrea Zsombok
Keyword(s):  
Copy Number ◽  
System Analysis ◽  
Renin Angiotensin System ◽  
Digital Pcr ◽  
Droplet Digital Pcr ◽  
Gene Copy ◽  
Angiotensin System ◽  
Renin Angiotensin ◽  
Copy Numbers ◽  
Intrarenal Ras

The intrarenal renin-angiotensin system (RAS) has been shown to play crucial roles in the development of hypertension and RAS associated kidney injury including diabetic nephropathy. Although some circulating RAS components are filtered into kidneys and contribute to the regulation of intrarenal RAS activity, evaluating expression levels of RAS components in the kidney is important to elucidate the mechanisms underlying intrarenal RAS activation. Digital PCR is a new technique that has been established to quantify absolute target gene levels, which allows for comparisons of different gene levels. Thus, this study was performed to establish profiles of absolute gene copy numbers for intrarenal RAS components in wild-type (WT) rats, WT and streptozotocin (STZ)-induced diabetic mice. Male Sprague-Dawley rats (N=5) and male C57BL/6J mice were used in this study. The mice were subjected to either control (N=5) or STZ (200 mg/kg, N=4) injection. Seven days after STZ injection, copy numbers of renal cortical angiotensinogen (AGT), angiotensin-converting enzyme (ACE), ACE2, angiotensin type 1 receptor a (AT1a), and AT2 mRNA were determined by a droplet digital PCR. Since (pro)renin proteins produced by juxtaglomerular cells are secreted to circulating system, analysis of renin mRNA was excluded from this evaluation. In the renal cortex of WT rats, the copy number of AGT was higher than other measured RAS components (AGT: 719.2±46.6, ACE: 116.0±14.9, ACE2: 183.6±21.5, AT1a: 196.0±25.2 copies in 1 ng total RNA). AT2 levels were lower than other components (0.068±0.01 copies). In WT mice, ACE exhibited the highest copy number in the components (AGT: 447.2±29.0, ACE: 1662.4±61.2, ACE2: 676.8±41.5, AT1a: 867.0±16.8, AT2: 0.049±0.01 copies). Although STZ-induced diabetes did not change ACE2 and AT1a, ACE levels were reduced (765.5±98.1 copies) and AT2 levels were augmented (0.10±0.01 copies) as previously demonstrated. Accordingly, the absolute quantification by digital PCR established precise gene profiles of intrarenal RAS components, which will provide rationales for targeting the each component in future studies. Furthermore, the results indicate that the high sensitive assay accurately quantifies rare target genes including intrarenal AT2.

scholarly journals Copy Number and Prevalence of Porcine Endogenous Retroviruses (PERVs) in German Wild Boars

Viruses ◽  
2020 ◽  
Vol 12 (4) ◽  
pp. 419
Author(s):  
Luise Krüger ◽  
Milena Stillfried ◽  
Carolin Prinz ◽  
Vanessa Schröder ◽  
Lena Katharina Neubert ◽  
...  
Keyword(s):  
Wild Boar ◽  
Copy Number ◽  
Digital Pcr ◽  
Endogenous Retroviruses ◽  
Wild Boars ◽  
Domestic Pigs ◽  
Copy Numbers ◽  
Cellular Genes ◽  
Porcine Endogenous Retroviruses ◽  
Different Populations

Porcine endogenous retroviruses (PERVs) are integrated in the genome of pigs and are transmitted like cellular genes from parents to the offspring. Whereas PERV-A and PERV-B are present in all pigs, PERV-C was found to be in many, but not all pigs. When PERV-C is present, recombination with PERV-A may happen and the PERV-A/C recombinants are characterized by a high replication rate. Until now, nothing has been known about the copy number of PERVs in wild boars and little is known about the prevalence of the phylogenetically youngest PERV-C in ancient wild boars. Here we investigated for the first time the copy number of PERVs in different populations of wild boars in and around Berlin using droplet digital PCR. Copy numbers between 3 and 69 per genome have been measured. A lower number but a higher variability was found compared to domestic pigs, including minipigs reported earlier (Fiebig et al., Xenotransplantation, 2018). The wild boar populations differed genetically and had been isolated during the existence of the Berlin wall. Despite this, the variations in copy number were larger in a single population compared to the differences between the populations. PERV-C was found in all 92 analyzed animals. Differences in the copy number of PERV in different organs of a single wild boar indicate that PERVs are also active in wild boars, replicating and infecting new cells as has been shown in domestic pigs.

scholarly journals Intragenic variants in the SMN1 gene determine the clinical phenotype in 5q spinal muscular atrophy

2020 ◽  
Vol 6 (5) ◽  
pp. e505
Author(s):  
Rodrigo de Holanda Mendonça ◽  
Ciro Matsui ◽  
Graziela Jorge Polido ◽  
André Macedo Serafim Silva ◽  
Leslie Kulikowski ◽  
...  
Keyword(s):  
Spinal Muscular Atrophy ◽  
Copy Number ◽  
Clinical Phenotype ◽  
Muscular Atrophy ◽  
Large Population ◽  
Survival Motor Neuron ◽  
Compound Heterozygous ◽  
Pathogenic Variants ◽  
Exon 1 ◽  
Compound Heterozygous Variants

ObjectiveThe aim of the study was to report the proportion of homozygous and compound heterozygous variants in the survival motor neuron 1 (SMN1) gene in a large population of patients with spinal muscular atrophy (SMA) and to correlate the severity of the disease with the presence of specific intragenic variants in SMN1 and with the SMN2 copy number.MethodsFour hundred fifty Brazilian patients with SMA were included in a retrospective study, and clinical data were analyzed compared with genetic data; the SMN2 copy number was obtained by multiplex ligation-dependent probe amplification and pathogenic variants in SMN1 by next-generation sequencing.ResultsFour hundred two patients (89.3%) presented homozygous exon 7-SMN1 deletion, and 48 (10.7%) were compound heterozygous for the common deletion in one allele and a point mutation in the other allele. Recurrent variants in exons 3 and 6 (c.460C>T, c.770_780dup and c.734_735insC) accounted for almost 80% of compound heterozygous patients. Another recurrent pathogenic variant was c.5C>G at exon 1. Patients with c.770_780dup and c.734_735insC had a clinical phenotype correlated with SMN2 copy number, whereas the variants c.460C>T and c.5C>G determined a milder phenotype independently of the SMN2 copies.ConclusionsPatients with specific pathogenic variants (c.460C>T and c.5C>G) presented a milder phenotype, and the SMN2 copy number did not correlate with disease severity in this group.

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

2019 ◽  
Vol 65 (9) ◽  
pp. 1153-1160 ◽  
Author(s):  
Kévin Cassinari ◽  
Olivier Quenez ◽  
Géraldine Joly-Hélas ◽  
Ludivine Beaussire ◽  
Nathalie Le Meur ◽  
...  
Keyword(s):  
Copy Number ◽  
Segregation Analysis ◽  
Genetic Diseases ◽  
Genomic Medicine ◽  
Digital Pcr ◽  
Copy Number Variations ◽  
Locked Nucleic Acid ◽  
Targeted Analysis ◽  
Cost Efficient ◽  
High Flexibility

Abstract BACKGROUND Rare copy number variations (CNVs) are a major cause of genetic diseases. Simple targeted methods are required for their confirmation and segregation analysis. We developed a simple and universal CNV assay based on digital PCR (dPCR) and universal locked nucleic acid (LNA) hydrolysis probes. METHODS We analyzed the mapping of the 90 LNA hydrolysis probes from the Roche Universal ProbeLibrary (UPL). For each CNV, selection of the optimal primers and LNA probe was almost automated; probes were reused across assays and each dPCR assay included the CNV amplicon and a reference amplicon. We assessed the assay performance on 93 small and large CNVs and performed a comparative cost-efficiency analysis. RESULTS UPL-LNA probes presented nearly 20000000 occurrences on the human genome and were homogeneously distributed with a mean interval of 156 bp. The assay accurately detected all the 93 CNVs, except one (<200 bp), with coefficient of variation <10%. The assay was more cost-efficient than all the other methods. CONCLUSIONS The universal dPCR CNV assay is simple, robust, and cost-efficient because it combines a straightforward design allowed by universal probes and end point PCR, the advantages of a relative quantification of the target to the reference within the same reaction, and the high flexibility of the LNA hydrolysis probes. This method should be a useful tool for genomic medicine, which requires simple methods for the interpretation and segregation analysis of genomic variations.

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