Discovery of New Players in Congenital Heart Defects Using Genome Wide Approaches - Mahdi Moradi Marjaneh


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Authors: Mahdi Moradi Marjaneh, Edwin P. Kirk, Peter C. Thomson, Tram B. Doan, Ian C.A. Martin, Chris Moran, Richard P. Harvey

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Discovery of New Players in Congenital Heart Defects Using Genome Wide Approaches - Mahdi Moradi Marjaneh

  1. 1. 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 1Victor 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:, Introduction • 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. Krasuski, 2007 Patent foramen ovale (PFO) Normal heart • 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. Objectives 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 intercross design. 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 fossa ovalis. LOD 10 FVL 8 FOW 6 • Foramen ovale width (FOW): The maximum width of the foramen ovale perpendicular to the FVL. 4 2 0 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. 0 1 2 100 3 4 200 5 6 7 300 8 9 400 10 11 12 13 14 15 600 16 17 18 700 19 Chr 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. FOW QTL Chr Impact Old_codon/New_codon 66449494 Mtap2 STOP_GAINED High CAG/TAG 66450672 Mtap2 STOP_GAINED High TGG/TGA 2 71100933 Dync1i2 STOP_GAINED High TGG/TGA 2 71715328 Pdk1 SPLICE_SITE_ACCEPTOR High 4 9549010 Asph STOP_LOST3 High TGA/TGG 4 9561371 Asph STOP_GAINED High TGG/TGA 4 41701766 Sigmar1 STOP_LOST High TGA/TTA 4 43950254 Reck STOP_GAINED High CAG/TAG 9 Figure 3. All QTL identified by F2 study (red) were confirmed and significantly narrowed using AIL (blue). Some F2 QTL resolved into multiple peaks. Gene_name Effect 1 FVL QTL Position 1 Advanced Intercross Line (AIL) 500 20248886 Zfp26 STOP_GAINED High CGA/TGA 9 44590278 Ift46 STOP_GAINED High CGA/TGA 9 44597761 Ift46 STOP_GAINED High GAA/TAA 13 14280024 Arid4b STOP_GAINED High CAA/TAA 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 protein sequence. Conclusions References • 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; 98(5):651-658.