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Detection of F8 int22h inversions using digital droplet PCR and mile-post assays
Kristianstad University, Faculty of Natural Science, Avdelningen för miljö- och biovetenskap. Kristianstad University, Faculty of Natural Science, Forskningsmiljön Biomedicin.
Kristianstad University, Faculty of Natural Science, Forskningsmiljön Biomedicin. Kristianstad University, Faculty of Natural Science, Avdelningen för miljö- och biovetenskap.
Lunds universitet.
Skåne University Hospital.
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2020 (English)In: Journal of Thrombosis and Haemostasis, ISSN 1538-7933, E-ISSN 1538-7836Article in journal (Refereed) Epub ahead of print
Abstract [en]

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 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 located 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 30-40% linked molecules for loci 12-15 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.

Place, publisher, year, edition, pages
2020.
Keywords [en]
Factor VIII, Genetic Linkage, Hemophilia A, Polymerase Chain Reaction, Sequence Inversion
Identifiers
URN: urn:nbn:se:hkr:diva-20381DOI: 10.1111/jth.14760PubMedID: 32031725OAI: oai:DiVA.org:hkr-20381DiVA, id: diva2:1397521
Available from: 2020-02-27 Created: 2020-02-27 Last updated: 2020-02-27Bibliographically approved

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Manderstedt, EricLind-Halldén, ChristinaHalldén, C
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