Discovery of New Players in Congenital Heart Defects Using Genome Wide Approaches - Mahdi Moradi Marjaneh
Discovery of New Players in Congenital Heart Defects
using Genome-wide Approaches
Mahdi Moradi Marjaneh1,2, Edwin P. Kirk2,3, Peter C. Thomson4, Tram B. Doan1, Ian C.A. Martin4, Chris Moran4, Richard P. Harvey1,5
Chang Cardiac Research Institute, Sydney, NSW, Australia
2School of Women and Children’s Health, University of New South Wales, Sydney, NSW, Australia
3Department of Medical Genetics, Sydney Children’s Hospital, NSW, Australia
4ReproGen - Animal Biosciences Group, University of Sydney, NSW, Australia
5St. Vincent’s Clinical School, University of New South Wales, Sydney, NSW, Australia
Email: firstname.lastname@example.org, email@example.com
• The interatrial septum in the mammalian heart is formed by the septum primum (blue) and the septum secundum (green) which overlap each other during
cardiac development and fuse together after birth.
• In 25% of the human population the two septa do not fuse completely after birth leading to an abnormal connection between the two atrial chambers termed
patent foramen ovale (PFO).
• PFO is clinically important since it is possibly associated with several major clinical conditions, in particular, stroke and migraine headaches.
Patent foramen ovale (PFO)
• Despite previous studies on the genetic basis of cardiac defects in humans and animal models, the genetic complexity of PFO has not been addressed in depth.
Identify genetic causation of PFO using genome-wide approaches:
• Establish quantitative parameters of atrial septum
• Identify quantitative trait loci (QTL) for the parameters
• Identify the causative gens by finding the variations between the parental lines under QTL peaks
Methodology and Results
2. QTL mapping of quantitative parameters using F2 intercross design
1. Establish quantitative parameters of atrial septum
A genome-wide QTL mapping of quantitative parameters (FVL and FOW) was performed using a mouse F2
Quantitative anatomical parameters of atrial septum were established in
mouse model. These parameters were strongly correlated to the risk of PFO
and therefore could be studied as proxies for the risk of PFO.
The parameters are as follows:1,2
The parental strains in our F2 intercross study were QSi5 and 129T2/SvEms which were selected based on
having extreme values for mean FVL and prevalence of PFO.1,2
• Flap valve length (FVL): The length of the flap valve from the edge of the
crescent (an embryonic landmark of atrial septum) to the distal rim of the
• Foramen ovale width (FOW): The maximum width of the foramen ovale
perpendicular to the FVL.
Figure 1. Atrial septal morphology in the mouse (left aspect)2
3. Confirm and fine map QTL using advanced intercross line (AIL)
We established an advanced intercross line (AIL) by continuous breeding of parental strains (QSi5 and
129T2/SvEms) for 14 generations. This would significantly increase the chance of crossing over between
any two loci and would result in a genetic map with higher resolution.
Figure2. Genome-wide QTL mapping identified 6 significant QTL (LOD > 4.3) for FOW and FVL.2
4. Identify the causative genes by finding variations between the parental
stains under the QTL peaks
While advanced intercross line (AIL) significantly narrowed the QTL region, each AIL QTL still harboured a
high number of candidate genes.
We performed QTL mapping for F14 generation.
We performed whole genome sequencing of the 2 parental strains to identify the variations between them.
The variations were filtered for those on QTL regions, being related to heart, and having high impact on
protein sequence resulted in 13 promising candidates.
Figure 3. All QTL identified by F2 study (red) were confirmed
and significantly narrowed using AIL (blue). Some F2 QTL
resolved into multiple peaks.
Advanced Intercross Line (AIL)
Table 1. Promising candidates for
the risk of patent foramen ovale
(PFO). The genotype of each
candidate differs between the
two parental strains. In addition,
each candidate is located on a
QTL region, expressed in the
heart, and has a high impact on
• Our results provide the first high-resolution picture of genetic complexity of atrial septal variation
and risk of PFO in a mouse model.
1. Biben C, Weber R, Kesteven S, Stanley E, McDonald L, Elliott DA, Barnett L, Köentgen F, Robb L, Feneley M, Harvey
RP. Cardiac septal and valvular dysmorphogenesis in mice heterozygous for mutations in the homeobox gene NKX25. Circ Res. 2000; 87(10):888-895.
• Combining QTL mapping with new genomic technologies made a major progress towards
identification of contributing genes.
2. Kirk EP, Hyun C, Thomson PC, Lai D, Castro ML, Biben C, Buckley MF, Martin IC, Moran C, Harvey RP. Quantitative
trait loci modifying cardiac atrial septal morphology and risk of patent foramen ovale in the mouse. Circ Res. 2006;