Genetic Origin of Congenital Heart Disease
Identification of genetic variants causing congenital heart disease

Congenital heart defects (CHD) constitute a major percentage of all clinically
significant birth defects. Recent studies show a high degree of heritability of
certain left ventricular outflow tract (LVOT) obstructions such as the bicuspid
aortic valve (BAV). Despite the numerous advanced therapies currently available
for a number of congenital heart defects, significant morbidity and mortality
are still associated with CHD. Furthermore it is still difficult to predict
the fetal and postnatal course of a CHD. New treatment strategies focus on long
term consequences of CHD such as neurological outcome and late complications.
However understanding the possible causes of CHD will permit insight into the
pathobiological basis of the congenital heart problem and allow definition of
disease risk, which are two critical elements in disease prevention.
It is very important to determine whether there is an underlying genetic basis
for the disease phenotype, for the following reasons: (1) There may be other
important organs involved in the disease phenotype. (2) Prognostic information
for clinical outcomes might be available. Understanding their genetic cause may
give insight in the pre- and postnatal course of the disease. The establishment
of a genotype-phenotype relation will personalize patient care. It may select
patients at risk for a complicated antenatal course who may benefit from a
fetal intervention. It may also help to predict postnatal disease progression.
Patients at risk for rapid progression of their disease may be followed more
closely, while for others less intense monitoring would suffice. (3) There may
be important genetic reproductive risks the family should know about. (4) There
may be other family members for whom genetic testing or regular preventive
diagnosis for early disease detection is advisable. (5) Once the causative gene
is discovered screening may postnatally be performed in cord blood, obviating
the need for postnatal evaluation and follow-up by a pediatric cardiologist.
(6) Determination of causative genes will be the first step towards a therapy
to cure CHD as demonstrated by the recent advances in the treatment of Marfan
syndrome.

Current conventional approaches for discovering monogenetic factors involved in
congenital heart defects include pedigree studies and candidate gene screens.
Unfortunately, this approach relies on large families exhibiting a Mendelian
pattern of inheritance. For CHD such cases are rare.
Candidate genes are typically selected based on pre-existing knowledge
originated from basic scientific research and/or clinical studies.
Unfortunately the candidate approach is restricted to studying *old friends*
genes previously suspected to be implicated in the disease, precluding the
possibility for discovering novel, unsuspected players.
In recent years novel high-throughput sequencing technologies, also known as
Next-generation sequencing (NGS) is set to alter the landscape of personalized
diagnostics, through the cataloging of SNVs, deletions, duplications, copy
number variations, and genomic rearrangements for the entire genome of a single
affected individual within a short period of time.
The zebrafish is a very cost effective in vivo model to test the number of
candidate genes that is expected from a whole genome project. Furthermore the
model has proven its use in human cardiovascular disease. Morpholino-based
knock-down technology in zebrafish embryos allows the inhibition of specific
gene activity during cardiac development. When combined with mRNA rescue using
the human wild-type versus the patient derived variant, this method allows for
a true validation of the variants influence on protein function.
NGS has widely proven its additional value in detecting de novo mutations in
CHD and the UMC Utrecht and the Hubrecht Institition have proven that they can
successfully apply NGS and perform gene expression studies.

Primary Objective: Identification of genetic variants causative of CHD.
Expression of candidate genes in a zebrafish model. Overall goal is to gain
insight in cardiac development by expanding our knowledge of genetic
components in de novo and familial CHD.

Secondary Objective(s):
- To determine the functional consequences of identified genetic variants in
CHD. Clinical information (type of CHD, clinical data and associated diseases)
will be compared with observed variants of candidate genes. Pathways involved
in the pathophysiology of CHD will be explored.

- When novel diagnostic genes are discovered they will be added to the next
version of the cardiome chip which is a diagnostic chip containing all genes
know to cause cardiac disease known to date. It has been developed by de
division of cardiology and medical genetics. This practical implementation of
our study will be done according to their diagnostic standards and internal
protocols.

All patients with CHD undergoing and invasive procedure (cardiac
catheterization of cardiac surgery) at the Wilhelmina Children*s Hospital, UMC
Utrecht, from 01-04-2015 onwards will be asked to participate in the study.
Broad consent will be obtained for biobanking patients blood and tissue.
Clinical data of all patients is available since this is the indication for the
invasive procedure.

All invasive cardiac procedures (cardiac catheterization or cardiac surgery)
are performed under general anaesthesia with central venous and/or arterial
access. Arterial and venous samples are routinely obtained during these
procedures. During one of the routine blood sample drawings some extra blood
will be obtained for DNA isolation. Blood samples are max 10 ml from
individuals of 5 years and older, 4-10 ml from children 1-5 years of age, and
2-5 ml from children < 1 year of age. Cardiac tissue or tissue of the great
vessels is removed in approximately 50% of all cardiac surgeries performed.
Instead of disposing this tissue, this tissue will be collected at the
operating theatre and stored in a bio bank. When tissue is not routinely
removed during surgery, tissue will not be obtained from the patient.

Clinical information will be collected from the patients* medical record.

Every month around 50 invasive procedures (20 cardiac catheterizations and 30
cardiac surgeries) are performed on children with CHD at the Wilhelmina
Children*s Hospital, UMC Utrecht. Bimonthly a team of experts consisting of at
least a pediatric cardiologist (department of pediatric cardiology (Breur)),
developmental biologist (Bakkers), a clinical geneticist (department of medical
genetics ((Baas)) and geneticist (division of genetics (van Haaften)) will
evaluate the around 100 children who underwent an invasive procedure and select
the cases that most likely have either sporadic CHD suggestive of a de novo
mutation or a familial case of CHD. These cases will progress to trio analyses
with NGS. Parents will be counselled by a clinical geneticist specialized in
CHD (Baas/vd Smagt). Genetic counselling will cover the risk of finding other
disease causing variants. Furthermore parents will be screened by an adult
cardiologist if familial CHD is suspected. Blood for DNA isolation will be
obtained from the parents. Separate consent for progressing to NGS will be
obtained.

The objective of the study is to identify genetic variants causative of CHD
with NGS.. Identified candidate genes will be studied for their role during
cardiac development. Expression of the candidate genes will be performed by in
situ hybridization techniques on zebrafish embryos.

The total duration of the study will be 2,5 years. After obtaining informed
consent, DNA from patients and family members will be collected in our
diagnostic DNA-lab followed by NGS. DNA and medical information will be stored
for a minimal of 15 years. The inclusion period will be from 01-06-2016 until
01-06-2018. Performance of NGS and expression studies will continue until
01-1-2019.

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