Droplet digital PCR and mile-post analysis for the detection of F8 int1h inversions

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Droplet digital PCR and mile-post analysis for the detection of F8 int1h inversions. / Manderstedt, Eric; Lind-Halldén, Christina; Ljung, Rolf; Astermark, Jan; Halldén, Christer.

I: Journal of Thrombosis and Haemostasis, Vol. 19, Nr. 3, 2021, s. 732-737.

Forskningsoutput: TidskriftsbidragArtikel i vetenskaplig tidskrift

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T1 - Droplet digital PCR and mile-post analysis for the detection of F8 int1h inversions

AU - Manderstedt, Eric

AU - Lind-Halldén, Christina

AU - Ljung, Rolf

AU - Astermark, Jan

AU - Halldén, Christer

N1 - This article is protected by copyright. All rights reserved.

PY - 2021

Y1 - 2021

N2 - BACKGROUND: F8 int1h inversions (Inv1) are detected in 1-2% of severe hemophilia A (HA) patients. Long-range polymerase chain reaction (PCR) and inverse-shifting PCR have been used to diagnose these inversions.OBJECTIVES: To design and validate a sensitive and robust assay for detection of F8 Inv1 inversions.METHODS: Archival DNA samples were investigated using mile-post assays and droplet digital PCR.RESULTS: Mile-post assays for Inv1 showing high specificities and sensitivities were designed and optimized. Analysis of four patients, two carrier mothers and 40 healthy controls showed concordance with known mutation status with one exception. One patient had a duplication involving exons 2-22 of the F8 gene instead of an Inv1 mutation. DNA mixtures with different proportions of wild type and Inv1 DNA correlated well with the observed relative linkage for both wild type and Inv1 assays and estimated the limit of detection of these assays to 2% of the rare chromosome.CONCLUSIONS: The mile-post strategy has several inherent control systems. The absolute counting of target molecules by both assays enables determination of template quantity, detection of copy number variants and rare variants occurring in primer and probe annealing sites and estimation of DNA integrity through the observed linkage. The presented Inv1 mile-post analysis offers sensitive and robust detection and quantification of the F8 int1h inversions and other rearrangements involving intron 1 in patients and their mothers.

AB - BACKGROUND: F8 int1h inversions (Inv1) are detected in 1-2% of severe hemophilia A (HA) patients. Long-range polymerase chain reaction (PCR) and inverse-shifting PCR have been used to diagnose these inversions.OBJECTIVES: To design and validate a sensitive and robust assay for detection of F8 Inv1 inversions.METHODS: Archival DNA samples were investigated using mile-post assays and droplet digital PCR.RESULTS: Mile-post assays for Inv1 showing high specificities and sensitivities were designed and optimized. Analysis of four patients, two carrier mothers and 40 healthy controls showed concordance with known mutation status with one exception. One patient had a duplication involving exons 2-22 of the F8 gene instead of an Inv1 mutation. DNA mixtures with different proportions of wild type and Inv1 DNA correlated well with the observed relative linkage for both wild type and Inv1 assays and estimated the limit of detection of these assays to 2% of the rare chromosome.CONCLUSIONS: The mile-post strategy has several inherent control systems. The absolute counting of target molecules by both assays enables determination of template quantity, detection of copy number variants and rare variants occurring in primer and probe annealing sites and estimation of DNA integrity through the observed linkage. The presented Inv1 mile-post analysis offers sensitive and robust detection and quantification of the F8 int1h inversions and other rearrangements involving intron 1 in patients and their mothers.

U2 - 10.1111/jth.15219

DO - 10.1111/jth.15219

M3 - Article

C2 - 33345381

VL - 19

SP - 732

EP - 737

JO - Journal of Thrombosis and Haemostasis

JF - Journal of Thrombosis and Haemostasis

SN - 1538-7933

IS - 3

ER -