Identification and functional characterization of causal genetic variants in patients with an unexplained bleeding tendency

Current screening and confirmatory laboratory tests lack sufficient sensitivity
for diagnosing all disturbances in the hemostatic system. Consequently, for
specific patients with an evident bleeding diathesis, a causal aberrance in
hemostasis cannot be identified. An important tool in the diagnostic workflow
for patients with unexplained bleeding tendency is Whole Exome Sequencing
(WES), which is used for identifying genetic variants in genes involved in
hemostasis. The current diagnostic approach for WES analysis in patients with a
bleeding diathesis in the Radboudumc consists of the HEMOS panel, a gene panel
consisting of 157 genes playing an important role in thrombosis and hemostasis.
HEMOS-restricted WES is followed by an unrestricted approach when HEMOS results
are negative. However, the diagnostic yield for both approaches is low, as a
causative variant was identified with the HEMOS panel in only 17% (20 out of
119) of patients, while the unrestricted approach has resulted in a diagnosis
in 1 out of 40 cases.

In this study we aim to re-analyze data of patients and families for whom the
original diagnostic WES analysis did not yield a causative variant in order to
identify new (putative) causative genes. Following identification of a
potentially causative variant we will perform follow up experiments to assess
the involvement of the variant in the patient*s phenotype.

Identification, segregation analysis, and functional characterization of
genetic variants possibly causing rare bleeding disorders.

The following questions will be answered:
1) Can we identify (new) causal variants that explain the phenotype of patients
with unexplained bleeding tendency?

Exploratory study of putative causal variants in the index cases as well as
affected and unaffected family members.

The individual patient and their family might benefit from the identification
of a causal variant as this may lead to a better understanding of the patient*s
(or families*) bleeding tendency. This may lead to validation for the patient
as their bleeding tendency would no longer be unexplained as well as possibly
improved prevention or treatment modalities.

There are some risks associated with participating in the study: blood
withdrawal is needed, both for in-depth diagnosis and analysis of the specific
bleeding tendency and for isolation of DNA for WES analysis. It is important to
note that some diagnostic approaches, specifically those that target
fibrinolysis, involve blood withdrawal after 10 minutes of stowing, and are
thus more invasive than regular blood withdrawal. Any blood withdrawal will
specifically be a burden for the unaffected family members, as these would not
have been subjected to an invasive procedure had they not participated in the
study. We have chosen to include unaffected family members because their
participation is vital for proving causality and excluding variants that do not
segregate with the phenotype.

WES analysis brings further, privacy-related burdens. Several steps are taken
to minimize, or eliminate the chance of secondary findings associated with WES.
A more detailed analysis of the ethical considerations and regulations taken
into account in designing the current study is found in Chapter 9 of
supplementary C1.