Detection of F8 int22h inversions using digital droplet PCR and mile‐post assays

Abstract

Background: Inversions involving intron 22 (Inv22) of F8 are detected in approximately 45% of all severe hemophilia A patients. Diagnosis is complicated by the large size of the ~9.5 kb int22h repeated sequence, which generates the inversions.Methods such as long-range polymerase chain reaction (PCR) and inverse-shifting PCR are currently used diagnostically, but suffer from low PCR efficiencies and are difficult to standardize. Objectives:To design and validate a sensitive and robust assay for the detection of F8 int22h inversions.Methods: Digital droplet PCR using mile-post assays was used to investigate archival DNA samples. Results:The detection of linkage as a function of physical distance between loci was investigated using an anchor locus and mile-post loci at 1, 6, 12 and 15 kb distances from the anchor locus. The proportion of linked molecules decreased with increasing distance between loci and showed 30% to 40% linked molecules for loci 12 to15 kb apart. Mile-post assays specific for wild type and Inv22 type 1 and 2 chromosomes were then designed and optimized. All three assays showed high specificities and sensitivities, with coefficients of variation <5% for all assays. Analysis of 106 patients and 20 carrier mothers showed complete concordance with previously known mutation status. The analysis demonstrated the robustness of the assays versus input DNA concentration (6 ng and higher) and level of fragmentation. Conclusions:Digital droplet PCR and mile-post assays can be used to detect F8 int22h inversions. The assay systems are technically simple to perform, highly efficient, and robust. K E Y W O R D Sfactor VIII, genetic linkage, hemophilia A, polymerase chain reaction, sequence inversion

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

“…We have previously reported a detection system for Inv22 inversions using milepost assays and ddPCR 5 . The present study reports an extension of this study including the design, optimization, and validation of technically simple milepost assays allowing for rapid and precise diagnosis of Inv1 inversions in both male patients and carrier mothers.…”

“…Droplet digital PCR used FAM and VIC dual-labeled probe systems to detect Inv1 target sequences in a similar way to what has been previously described for Inv22 target sequences. 5 In short, probes were designed using RealTimeDesign Software, BioSearch Technology (https://www.biose archt ech.com/displ ay.aspx?pagei d=54) and ordered from DNA Technology A/S. Digital PCR was performed using a QX100 ddPCR system from Bio-Rad.…”

“…We have previously reported a detection system for Inv22 inversions using milepost assays and ddPCR. 5 We would like to argue that the use of milepost assays for both types of F8 inversions allows for much more powerful detection of these inversions and will make standardization and cross-laboratory evaluation of these analyses easier. Furthermore, the presence of milepost systems for the detection of both types of inversions increases the clinical usefulness of these systems because both types of inversions can now be analyzed using the same platform.…”

“…Recently, droplet digital PCR (ddPCR) using a milepost strategy was described for the detection of intron 22 inversions 5 . The ddPCR produces water‐in‐oil emulsions that partitions the template DNA into thousands of small droplets.…”

“…4 Recently, droplet digital PCR (ddPCR) using a milepost strategy was described for the detection of intron 22 inversions. 5 The ddPCR produces water-in-oil emulsions that partitions the template DNA into thousands of small droplets. The presence or absence of one or several target molecules are then determined for individual droplets using flow cytometry.…”