Pezeshkpoor, Behnaz: Investigation of new mechanisms causing hemophilia A. - Bonn, 2014. - Dissertation, Rheinische Friedrich-Wilhelms-Universität Bonn.
Online-Ausgabe in bonndoc:
author = {{Behnaz Pezeshkpoor}},
title = {Investigation of new mechanisms causing hemophilia A},
school = {Rheinische Friedrich-Wilhelms-Universität Bonn},
year = 2014,
month = feb,

note = {The main aim of this work was identification of novel molecular mechanisms causing hemophilia A, with a special focus on patients without detectable mutation. In most of hemophilia A patients, mutations could be identified in the coding sequence of F8. However, despite tremendous improvements in mutation screening techniques, in a small number of patients no mutation is found. The focus of this work was the analysis of changes in regions of F8 that are usually being missed. These variations involve regions that are not assessed by current routine techniques.
To address all difficulties in finding the causal mutation, an extended diagnosis guideline is needed to reduce the number of cases classified as mutation negative hemophilia A patients. Therefore, in this study a practical experimental protocol was designed. Using this systematic screening method, the whole F8 locus was screened for novel genetic abnormalities and alternations. This protocol combined several mutation screening tools. Multiple ligation-dependent probe amplification and comparative genomic hybridization were used to detect copy number variations. Next generation sequencing (NGS) was applied subsequently in combination with long range (LR-) PCRs to re-sequence the whole genomic region of F8 in all patients. The LR-PCR approach identified two novel breakpoints. Moreover, after NGS, several unique deep intronic variations were found. Later, mRNA analysis was done to prove the causality of these intronic variations.
Using this experimental strategy, causative mutations in most of the patients were found and characterized. Interestingly, all causal mutations were within the F8 locus. The results of this work show once more the variety and complexity of mutations causing hemophilia A. Considering all the findings of this study complex rearrangements are the fundamental cause of number of severe hemophilia A cases. Moreover, the data presented here show the association of deep intronic mutations with hemophilia A phenotype. To diminish the number of mutation negative patients, there is a need to apply several techniques. Based on the results of this work, an extended diagnosis flowchart is proposed. This flowchart demonstrates techniques for successful identification and confirmation of novel mutations in hemophilia A patients and could serve as a model for genetic analysis of other monogenic diseases, where no pathogenic mutation is found.},

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