Analyzing Copy Number Variation with Droplet Digital PCR

Bell, Avery Davis; Usher, Christina L.; McCarroll, Steven A.

doi:10.1007/978-1-4939-7778-9_9

Cited by 50 publications

(39 citation statements)

References 16 publications

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“…When WES was performed in other family members, the inheritance of putative CNVs was also determined. Putative CNVs were validated using ddPCR 38 . Specifically, primers and probes were designed to amplify exons with putative deletions.…”

Section: Copy Number Variant Identification and Validationmentioning

confidence: 99%

The Genetic Landscape of Diamond-Blackfan Anemia

Ulirsch

Verboon

Kazerounian

et al. 2018

Preprint

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Diamond-Blackfan anemia (DBA) is a rare bone marrow failure disorder that affects 1 in 100,000 to 200,000 live births and has been associated with mutations in components of the ribosome. In order to characterize the genetic landscape of this genetically heterogeneous disorder, we recruited a cohort of 472 individuals with a clinical diagnosis of DBA and performed whole exome sequencing (WES). Overall, we identified rare and predicted damaging mutations in likely causal genes for 78% of individuals. The majority of mutations were singletons, absent from population databases, predicted to cause loss of function, and in one of 19 previously reported genes encoding for a diverse set of ribosomal proteins (RPs). Using WES exon coverage estimates, we were able to identify and validate 31 deletions in DBA associated genes. We also observed an enrichment for extended splice site mutations and validated the diverse effects of these mutations using RNA sequencing in patientderived cell lines. Leveraging the size of our cohort, we observed several robust genotype-phenotype associations with congenital abnormalities and treatment outcomes. In addition to comprehensively identifying mutations in known genes, we further identified rare mutations in 7 previously unreported RP genes that may cause DBA. We also identified several distinct disorders that appear to phenocopy DBA, including 9 individuals with biallelic CECR1 mutations that result in deficiency of ADA2. However, no new genes were identified at exome-wide significance, suggesting that there are no unidentified genes containing mutations readily identified by WES that explain > 5% of DBA cases. Overall, this comprehensive report should not only inform clinical practice for DBA patients, but also the design and analysis of future rare variant studies for heterogeneous Mendelian disorders.

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Section: Copy Number Variant Identification and Validationmentioning

confidence: 99%

The Genetic Landscape of Diamond-Blackfan Anemia

Ulirsch

Verboon

Kazerounian

et al. 2018

Preprint

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“…To examine the copy number of chromosome Y in the samples with possible mLOY, we performed droplet digital PCR . Genomic DNA samples were analyzed using the QX200 system (Bio‐Rad Laboratories).…”

Section: Methodsmentioning

confidence: 99%

“…To examine the copy number of chromosome Y in the samples with possible mLOY, we performed droplet digital PCR. 14…”

Section: Detection Of Mloy By Droplet Digital Pcrmentioning

confidence: 99%

Copy‐number analysis of Y‐linked loci in young men with non‐obstructive azoospermia: Implications for the rarity of early onset mosaic loss of chromosome Y

Suzuki

Kobori

Katsumi

et al. 2020

Reprod Medicine & Biology

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Purpose Mosaic loss of chromosome Y (mLOY) is a common feature in elderly men. If mLOY can also occur in young men, it may lead to spermatogenic failure due to loss of spermatogenic genes. Indeed, previous studies detected the 45,X/46,XY karyotype in a few young men with spermatogenic failure. The present study aimed to clarify the frequency of cryptic mLOY in reproductive‐aged men with spermatogenic failure. Methods We studied 198 men at ages 24‐55 years who presented with etiology‐unknown non‐obstructive azoospermia. Prior this study, these patients underwent G‐banding analysis for 20 leukocytes and were found to have 46,XY karyotype. We analyzed copy numbers of chromosome Y in blood cells by using semi‐quantitative multiplex PCR for AMELY/AMELX, array‐based comparative genomic hybridization (CGH) for the AMELY locus, and droplet digital PCR for SRY, USP9Y, and UTY. Results Multiplex PCR showed borderline low AMELY/AMELX ratios in three patients. However, for the three patients, CGH excluded deletion of the AMELY locus, and droplet digital PCR suggested preserved copy numbers of all tested loci. Conclusion This study highlights the rarity of leukocyte mLOY in reproductive‐aged men with spermatogenic failure. In addition, our data imply that standard karyotyping is sufficient to screen early onset mLOY.

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“…Droplet digital PCR (ddPCR) has emerged as a recent technology which allows for more precise quantification and analysis of DNA and RNA as compared to older PCR techniques such as real-time PCR [10]. Applications of ddPCR include assessment of gene copy number variation, vector titer and rare event detection [11][12][13]. It has been used to measure the number of copies of retroviral vectors integrated into induced pluripotent stem (iPS) cells [14] and CD34+ hematopoietic stem cells [15].…”

Section: Introductionmentioning

confidence: 99%

Application of droplet digital PCR for the detection of vector copy number in clinical CAR/TCR T cell products

Liu

Shi

et al. 2020

J Transl Med

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Background: Genetically engineered T cells have become an important therapy for B-cell malignancies. Measuring the efficiency of vector integration into the T cell genome is important for assessing the potency and safety of these cancer immunotherapies. Methods: A digital droplet polymerase chain reaction (ddPCR) assay was developed and evaluated for assessing the average number of lenti-and retroviral vectors integrated into Chimeric Antigen Receptor (CAR) and T Cell Receptor (TCR)-engineered T cells. Results: The ddPCR assay consistently measured the concentration of an empty vector in solution and the average number of CAR and TCR vectors integrated into T cell populations. There was a linear relationship between the average vector copy number per cell measured by ddPCR and the proportion of cells transduced as measured by flow cytometry. Similar vector copy number measurements were obtained by different staff using the ddPCR assay, highlighting the assays reproducibility among technicians. Analysis of fresh and cryopreserved CAR T and TCR engineered T cells yielded similar results. Conclusions: ddPCR is a robust tool for accurate quantitation of average vector copy number in CAR and TCR engineered T cells. The assay is also applicable to other types of genetically engineered cells including Natural Killer cells and hematopoietic stem cells.

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Analyzing Copy Number Variation with Droplet Digital PCR

Cited by 50 publications

References 16 publications

The Genetic Landscape of Diamond-Blackfan Anemia

The Genetic Landscape of Diamond-Blackfan Anemia

Copy‐number analysis of Y‐linked loci in young men with non‐obstructive azoospermia: Implications for the rarity of early onset mosaic loss of chromosome Y

Application of droplet digital PCR for the detection of vector copy number in clinical CAR/TCR T cell products

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