Copy Number Variation Analysis by Droplet Digital PCR

Härmälä, Suvi; Butcher, Robert

doi:10.1007/978-1-4939-7231-9_9

Cited by 25 publications

(15 citation statements)

References 19 publications

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“…Given the observed high frequency of PARK2 CNVs in this familial PD cohort (~5%), and the high cost of clinical-grade aCGH, we examined the feasibility of exon-by-exon ddPCR assay to detect PARK2 CNVs as a proof of principle. ddPCR is an emerging cost-effective method for sensitive, and reliable assessment of specific CNVs 36,37 . Indeed, all PARK2 CNVs described (subjects 6, 11, 20, 21 and 22) were robustly detected using ddPCR ( Figure 4A).…”

Section: Digital Droplet Pcr (Ddpcr)mentioning

confidence: 99%

“…36 Compared to standard quantitative PCR, digital PCR offers enhanced copy number and gene dosage sensitivity, precision, and reliability due to sample partitioning. 37 We explored the genetic molecular diagnostic rate for integrated ES and aCGH in a familial PD cohort. In addition, we evaluate ddPCR for confirmation of pathogenic CNVs, and using breakpoint sequencing, we elucidate potential mechanisms for CNV formation.…”

Section: Introductionmentioning

confidence: 99%

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Integrated Sequencing & Array Comparative Genomic Hybridization in Familial Parkinson’s Disease

Robak

Du²,

Yuan³

et al. 2019

Preprint

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Background: Parkinson's disease (PD) is a genetically heterogeneous condition; both single nucleotide variants (SNVs) and copy number variants (CNVs) are important genetic risk factors.We examined the utility of combining exome sequencing and genome-wide array-based comparative genomic hybridization (aCGH) for identification of PD genetic risk factors. Methods:We performed exome sequencing on 110 subjects with PD and a positive family history; 99 subjects were also evaluated using genome-wide aCGH. We interrogated exome sequencing and array comparative genomic hybridization data for pathogenic SNVs and CNVs at Mendelian PD gene loci. SNVs were confirmed via Sanger sequencing. CNVs were confirmed with custom-designed high-density aCGH, droplet digital PCR, and breakpoint sequencing. Results:Using exome sequencing, we discovered individuals with known pathogenic single nucleotide variants in GBA (p.E365K, p.T408M, p.N409S, p.L483P) and LRRK2 (p.R1441G and p.G2019S). Two subjects were each double heterozygotes for variants in GBA and LRRK2.Based on aCGH, we additionally discovered cases with an SNCA duplication and heterozygous intragenic GBA deletion. Five additional subjects harbored both SNVs (p.N52fs, p.T240M, p.P437L, p.W453*) and likely disrupting CNVs at the PARK2 locus, consistent with compound heterozygosity. In nearly all cases, breakpoint sequencing revealed microhomology, a mutational signature consistent with CNV formation due to DNA replication errors. Conclusions:Integrated exome sequencing and aCGH yielded a genetic diagnosis in 19.3% of our familial PD cohort. Our analyses highlight potential mechanisms for SNCA and PARK2 CNV formation, uncover multilocus pathogenic variation, and identify novel SNVs and CNVs for further investigation as potential PD risk alleles.

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Section: Digital Droplet Pcr (Ddpcr)mentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

Integrated Sequencing & Array Comparative Genomic Hybridization in Familial Parkinson’s Disease

Robak

Du²,

Yuan³

et al. 2019

Preprint

View full text Add to dashboard Cite

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“…Compared with standard quantitative PCR, digital PCR offers enhanced copy number and gene dosage sensitivity, precision, and reliability due to sample partitioning. 16 In addition, mechanisms of CNV formation in PD remain understudied.…”

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confidence: 99%

Integrated sequencing and array comparative genomic hybridization in familial Parkinson disease

Robak

Yuan

et al. 2020

Neurol Genet

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ObjectiveTo determine how single nucleotide variants (SNVs) and copy number variants (CNVs) contribute to molecular diagnosis in familial Parkinson disease (PD), we integrated exome sequencing (ES) and genome-wide array-based comparative genomic hybridization (aCGH) and further probed CNV structure to reveal mutational mechanisms.MethodsWe performed ES on 110 subjects with PD and a positive family history; 99 subjects were also evaluated using genome-wide aCGH. We interrogated ES and aCGH data for pathogenic SNVs and CNVs at Mendelian PD gene loci. We confirmed SNVs via Sanger sequencing and further characterized CNVs with custom-designed high-density aCGH, droplet digital PCR, and breakpoint sequencing.ResultsUsing ES, we discovered individuals with known pathogenic SNVs in GBA (p.Glu365Lys, p.Thr408Met, p.Asn409Ser, and p.Leu483Pro) and LRRK2 (p.Arg1441Gly and p.Gly2019Ser). Two subjects were each double heterozygotes for variants in GBA and LRRK2. Based on aCGH, we additionally discovered cases with an SNCA duplication and heterozygous intragenic GBA deletion. Five additional subjects harbored both SNVs (p.Asn52Metfs*29, p.Thr240Met, p.Pro437Leu, and p.Trp453*) and likely disrupting CNVs at the PRKN locus, consistent with compound heterozygosity. In nearly all cases, breakpoint sequencing revealed microhomology, a mutational signature consistent with CNV formation due to DNA replication errors.ConclusionsIntegrated ES and aCGH yielded a genetic diagnosis in 19.3% of our familial PD cohort. Our analyses highlight potential mechanisms for SNCA and PRKN CNV formation, uncover multilocus pathogenic variation, and identify novel SNVs and CNVs for further investigation as potential PD risk alleles.

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“…This method partitions a conventional qPCR into water-in-oil droplets numbering up to 20,000, which permits the amplification of a single-template molecule in each droplet [ 16 ]. PCR-positive and PCR-negative droplets are counted at the end of the amplification procedure, thereby providing direct and absolute quantification of target DNA in a digital format [ 15 23 24 ]. The fact that ddPCR does not require a reference or a standard calibrator curve for quantification is one of its major advantages over qPCR [ 15 25 26 ].…”

Section: Discussionmentioning

confidence: 99%

A novel urinary mRNA signature using the droplet digital polymerase chain reaction platform improves discrimination between prostate cancer and benign prostatic hyperplasia within the prostate-specific antigen gray zone

et al. 2020

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The aim of this study was to identify a noninvasive urinary marker for prostate cancer (PCa) diagnosis and to validate the clinical performance of this novel urinary mRNA signature using the droplet digital polymerase chain reaction (ddPCR) approach. Materials and Methods: A gene expression microarray (HT-12, Illumina Inc., USA) was used to identify genes differentially expressed between 16 PCa and 8 benign prostatic hyperplasia (BPH) tissues; ddPCR (QX200; Bio-Rad Laboratories, USA) was carried out to quantify the expression of selected genes in urine. The urinary molecular PCa risk score (UMPCaRS) was calculated by using the sum of three upregulated genes as the numerator and the sum of three downregulated genes as the denominator. The diagnostic utility of the UMPCaRS was validated by using a screening set (10 PCa and 10 BPH samples) and a validation set (131 PCa and 105 BPH samples). Results: Three upregulated genes (PDLIM5, GDF-15, THBS4) and three downregulated genes (UPK1A, SSTR3, NPFFR2) were selected from the microarray and subjected to ddPCR. The UMPCaRS for PCa in the screening and validation sets was significantly higher than that for BPH. For the validation set, the diagnostic accuracy of the UMPCaRS was comparable with that of prostate-specific antigen (PSA). Importantly, in the "PSA gray zone" (3-10 ng/mL), the AUC for the UMPCaRS was 0.843 and that for PSA was 0.628 (p<0.001). Conclusions: The data demonstrate that the UMPCaRS is useful for discriminating between PCa and BPH in the "PSA gray zone".

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Copy Number Variation Analysis by Droplet Digital PCR

Cited by 25 publications

References 19 publications

Integrated Sequencing & Array Comparative Genomic Hybridization in Familial Parkinson’s Disease

Integrated Sequencing & Array Comparative Genomic Hybridization in Familial Parkinson’s Disease

Integrated sequencing and array comparative genomic hybridization in familial Parkinson disease

A novel urinary mRNA signature using the droplet digital polymerase chain reaction platform improves discrimination between prostate cancer and benign prostatic hyperplasia within the prostate-specific antigen gray zone

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