Introduction: For individuals with hemophilia, mutational analysis could represent a novel method of diagnosis. In addition, mutational analysis ideally would be available to better predict disease manifestations such as inhibitor development and to provide family planning to female carriers. However, many patients do not undergo testing due to high cost and lack of insurance reimbursement. Additionally, the time to receive sequencing results can be several weeks. We are developing a sequencing assay which can identify genetic mutations in the factor 8 and 9 genes using dried blood spots on filter paper with the goal of significantly lowering the turnaround time and cost of testing.

Methods: This is a single center, prospective, pilot study. Thus far, we have isolated DNA from blood spots on filter paper for sample preparation. DNA isolation is automated utilizing a Chemagen MSM1 robot (Perkin Elmer); which allows for the extraction of 96 samples at once in approximately 4 hours total time. Library preparation is automated with a NGS Janus Express (Perkin Elmer). The Nextera rapid capture and enrichment work flow is used to prepare samples for next generation sequencing, which is completed on a Miseq instrument (Illumina).

Results: We have successfully generated sequencing libraries from samples extracted from blood spots and they are indistinguishable from libraries generated from DNA isolated from whole blood. Using a previously designed targeted panel, we have successfully sequenced and analyzed patients with known F8 and F9 variants within one week of sample receipt. Currently we are able to detect sequence variants and insertions and deletion events up to 40 base pairs. We successfully identified causative mutations in 7 out of 8 individuals with known hemophilia (both A and B). All factor 9 mutations were identified successfully. Point mutations of the factor 8 gene were identified. Of note, a point mutation in a female carrier which was also seen her brother was successfully sequenced. Further method development is underway to detect the known factor 8 inversions from the sequencing data, providing a rapid single molecular test for both Factor 8 and Factor 9 patients.

Conclusion: DNA can successfully be extracted and analyzed for mutations in factor 8 and 9 genes from known hemophilia patients using dried blood spots. Once we have finalized the process for identifying inversions, we will compare results from 24 individuals known to be affected with hemophilia (16 hemophilia A, 8 hemophilia B) to 24 banked control DNA samples to provide proof of principle. Subsequently, we plan to expand the project to a larger clinical trial.

Table 1:
Table 1:. Initial Sequencing Results
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Initial Sequencing Results

Table 1:
Table 1:. Initial Sequencing Results
View largeDownload slide

Initial Sequencing Results

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Disclosures

Carpenter:Novo Nordisk: Honoraria, Membership on an entity's Board of Directors or advisory committees; Pfizer: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; CSL Behring: Honoraria, Membership on an entity's Board of Directors or advisory committees. Kingsmore:Illumina: Speakers Bureau.

Topics:
blood spot specimen, hemophilia a, mutation analysis, dna, massively-parallel genome sequencing, hemophilia b, molecular diagnostic techniques, family planning, whole blood, workflow

Author notes

*

Asterisk with author names denotes non-ASH members.

© 2014 by The American Society of Hematology
2014
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