Use of ubiquitous, highly heterozygous copy number variants and digital droplet polymerase chain reaction to monitor chimerism after allogeneic haematopoietic stem cell transplantation

Whitlam, John; Ling, Ling; Swain, Michael; Harrington, Tom; Mirochnik, Oksana; Brooks, Ian; Cronin, Sara; Challis, Jackie; Petrovic, Vida; Bruno, Damien L.; Méchinaud, Françoise; Conyers, Rachel; Slater, Howard R.

doi:10.1016/j.exphem.2017.01.004

Cited by 6 publications

(15 citation statements)

References 13 publications

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“…The informativity of interrogating various numbers of deletion copy number loci has been previously modeled(11,12) or experimentally evaluated(12,13). We empirically determined the informativity of our panel for eight unrelated donors, and for two parents and ten siblings of a CEPH Reference Family in order to model transplantation occurring between first-degree relatives (Supplemental Tables 2 and 3).…”

Section: Resultsmentioning

confidence: 99%

“…smMIPs were designed against 40 deletion copy number variants found in high frequencies across populations (13,25), supplemented with three additional polymorphic loci ( UGT2B17 , chr2:52751531–52782353, and chr8:39251030–39372444)(11), and two sex-linked loci ( F9 and SRY ). Design of probes optimized for uniqueness of binding sites and optimal guanine-cytosine content was performed against hg19 using the program MIPgen(26) as previously (27)to tile across each region.…”

Section: Methodsmentioning

confidence: 99%

“…The most common clinical methods type polymorphic short tandem repeat markers, which have a limit of sensitivity approaching 1% (10). We (11) and subsequently others (12,13) more recently developed methods to interrogate deletion copy number variants, a sub-class of copy number variation representing genomic regions tens to hundreds of thousands of base pairs long that are present in some individuals but absent in others (14–17). The first application of these markers utilized microscopic examination of cells subjected to FISH for informative loci (11), whereas later, real-time PCR (RT-PCR)(12) and digital droplet PCR (ddPCR) (13) assays offered advantages in target multiplexing and sensitivity.…”

Section: Introductionmentioning

confidence: 99%

“…We (11) and subsequently others (12,13) more recently developed methods to interrogate deletion copy number variants, a sub-class of copy number variation representing genomic regions tens to hundreds of thousands of base pairs long that are present in some individuals but absent in others (14–17). The first application of these markers utilized microscopic examination of cells subjected to FISH for informative loci (11), whereas later, real-time PCR (RT-PCR)(12) and digital droplet PCR (ddPCR) (13) assays offered advantages in target multiplexing and sensitivity. Nevertheless, even ddPCR chimerism assays have finite sensitivities (1 in 2,000 cells, or 0.05%(13)) and lesser limits of quantification (accuracy to 0.5% chimerism)(13), while interrogating only a limited number of loci due to restrictions with primer multiplexing and requiring specialized instrumentation (18).…”

Section: Introductionmentioning

confidence: 99%

“…The first application of these markers utilized microscopic examination of cells subjected to FISH for informative loci (11), whereas later, real-time PCR (RT-PCR)(12) and digital droplet PCR (ddPCR) (13) assays offered advantages in target multiplexing and sensitivity. Nevertheless, even ddPCR chimerism assays have finite sensitivities (1 in 2,000 cells, or 0.05%(13)) and lesser limits of quantification (accuracy to 0.5% chimerism)(13), while interrogating only a limited number of loci due to restrictions with primer multiplexing and requiring specialized instrumentation (18). …”

Section: Introductionmentioning

confidence: 99%

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Ultrasensitive Detection of Chimerism by Single-Molecule Molecular Inversion Probe Capture and High-Throughput Sequencing of Copy Number Deletion Polymorphisms

Waalkes

Penewit

et al. 2018

Clinical Chemistry

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BACKGROUND: Genomic chimerism, the co-occurrence of cells from different genetic origins, provides important diagnostic information in diverse clinical contexts, including graft injury detection and longitudinal surveillance of hematopoietic stem cell transplantation patients, but existing assays are limiting. Here we applied single-molecule molecular inversion probes (smMIPs), a high throughput sequencing technology combining multiplexed target capture with read quantitation mediated by unique molecular identifiers, to detect chimerism based on the presence or absence of polymorphic genomic loci. METHODS: We designed a 159 smMIP panel targeting 40 autosomal regions of frequent homozygous deletion across human populations and two sex-linked loci. We developed methods for detecting and quantitating loci absent from one cell population but present in another, which could be used to sensitively identify chimeric cell populations. RESULTS: Unrelated individuals and first-degree relatives were highly polymorphic across the loci examined. Using synthetic DNA mixtures, limits of detection of at least 1 in 10,000 chimeric cells was demonstrated without prior knowledge of genotypes, and mixtures of up to four separate donors could be deconvoluted. Quantitative linearity over four orders of magnitude and false positive rates below 1 in 85,000 events were achieved. 11 of 11 post-transplant clinical specimens from patients with hematological malignancies testing positive for residual cancer by conventional methods had detectable chimeric populations by smMIP, while 11 of 11 specimens testing negative by conventional methods were low-positive for chimerism by smMIP. CONCLUSIONS: smMIPs are scalable to high sensitivity and large numbers of informative markers, enabling ultrasensitive chimerism detection for many clinical purposes.

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Section: Resultsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

See 3 more Smart Citations

Ultrasensitive Detection of Chimerism by Single-Molecule Molecular Inversion Probe Capture and High-Throughput Sequencing of Copy Number Deletion Polymorphisms

Waalkes

Penewit

et al. 2018

Clinical Chemistry

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Ultrasensitive Quantitation of Genomic Chimerism by Single-Molecule Molecular Inversion Probe Capture and High-Throughput Sequencing of Copy Number Deletion Polymorphisms

Wu¹,

Kanaan²,

Penewit³

et al. 2022

The Journal of Molecular Diagnostics

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Understanding Donor-derived Cell-free DNA in Kidney Transplantation: An Overview and Case-based Guide for Clinicians

et al. 2022

Self Cite

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Kidney transplant recipients undergo lifelong monitoring of allograft function and evaluation for transplant complications. The current monitoring paradigm utilizes blood, urine, and tissue markers that are insensitive, nonspecific, or invasive to obtain. As a result, problems are detected late, after significant damage has accrued, and often beyond the time at which complete resolution is possible. Indeed, most kidney transplants eventually fail, usually because of chronic rejection and other undetected injury. There is a clear need for a transplant-specific biomarker that enables a proactive approach to monitoring via early detection of reversible pathology. A biomarker that supports timely and personalized treatment would assist in achieving the ultimate goal of improving allograft survival and limiting therapeutic toxicity to the recipient. Donor-derived cell-free DNA (ddcfDNA) has been proposed as one such transplant biomarker. Although the test is presently utilized most in the United States, it is conceivable that its use will become more widespread. This review covers aspects of ddcfDNA that support informed use of the test by general nephrologists, including the basic biology of ddcfDNA, methodological nuances of testing, and general recommendations for use in the kidney transplant population. Clinical contexts are used to illustrate evidence-supported interpretation of ddcfDNA results and subsequent management. Finally, knowledge gaps and areas for further study are discussed.

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Use of ubiquitous, highly heterozygous copy number variants and digital droplet polymerase chain reaction to monitor chimerism after allogeneic haematopoietic stem cell transplantation

Cited by 6 publications

References 13 publications

Ultrasensitive Detection of Chimerism by Single-Molecule Molecular Inversion Probe Capture and High-Throughput Sequencing of Copy Number Deletion Polymorphisms

Ultrasensitive Detection of Chimerism by Single-Molecule Molecular Inversion Probe Capture and High-Throughput Sequencing of Copy Number Deletion Polymorphisms

Ultrasensitive Quantitation of Genomic Chimerism by Single-Molecule Molecular Inversion Probe Capture and High-Throughput Sequencing of Copy Number Deletion Polymorphisms

Understanding Donor-derived Cell-free DNA in Kidney Transplantation: An Overview and Case-based Guide for Clinicians

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