Over-expression of DSCAM and COL6A2 cooperatively generates congenital heart defects

Abstract

A significant current challenge in human genetics is the identification of interacting genetic loci mediating complex polygenic disorders. One of the best characterized polygenic diseases is Down syndrome (DS), which results from an extra copy of part or all of chromosome 21. A short interval near the distal tip of chromosome 21 contributes to congenital heart defects (CHD), and a variety of indirect genetic evidence suggests that multiple candidate genes in this region may contribute to this phenotype. We devised a tiered genetic approach to identify interacting CHD candidate genes. We first used the well vetted Drosophila heart as an assay to identify interacting CHD candidate genes by expressing them alone and in all possible pairwise combinations and testing for effects on rhythmicity or heart failure following stress. This comprehensive analysis identified DSCAM and COL6A2 as the most strongly interacting pair of genes. We then over-expressed these two genes alone or in combination in the mouse heart. While over-expression of either gene alone did not affect viability and had little or no effect on heart physiology or morphology, co-expression of the two genes resulted in ≈50% mortality and severe physiological and morphological defects, including atrial septal defects and cardiac hypertrophy. Cooperative interactions between DSCAM and COL6A2 were also observed in the H9C2 cardiac cell line and transcriptional analysis of this interaction points to genes involved in adhesion and cardiac hypertrophy. Our success in defining a cooperative interaction between DSCAM and COL6A2 suggests that the multi-tiered genetic approach we have taken involving human mapping data, comprehensive combinatorial screening in Drosophila, and validation in vivo in mice and in mammalian cells lines should be applicable to identifying specific loci mediating a broad variety of other polygenic disorders.

Figure 1. Survey of heart defects caused by over-expression of CHD candidate genes in the fly.

a) Scheme depicting how candidate CHD genes from the distal region of chromosome 21 were screened by expressing them in the fly heart individually and in all possible pair-wise combinations. b) Electrical pacing heart performance stress chamber (left). Enlarged view of flies in pacing chamber (right). c) Example of increased heart failure rate in flies mis-expressing particular combinations of CHD candidate genes in the heart. The heart failure phenotype is presented as the percentage of flies whose heart fibrillated or stopped immediately following the pacing regimen (N = 200). (*Chi square p<0.05 for DSCAM+ COL6A2 and DSCAM+ SH3BGR). d) Interaction grid summarizing interactions between CHD candidate genes in the fly heart resulting in particular cardiac phenotypes. H  =  Heart rate, F  =  failure rate (% of flies that exhibited heart asystole or fibrillation immediately following the electrical pacing regime) and R  =  recovery rate (% of flies that exhibited recovered heart rate 2 minutes after the end of electrical pacing). Colored backgrounds indicate significant difference in heart function relative to non-expressing UAS controls (Chi square, p<0.05). Blue  =  heart rate; Red  =  stress-induced failure rate; Yellow  =  recovery rate following heart failure. e) Example of a genetic interaction between the CHD candidate genes SH3BGR and COL6A1 in the fly eye using the GMR-GAL4 driver. f) Summary of all genetic interactions between CHD candidate genes in the fly heart (lower triangles in each interaction box) and eye (upper triangles in each interaction box). Blue  =  no detectable interaction, orange  =  moderate interaction and red  =  strong interaction. The combination exhibiting the strongest interactions in both the heart and eye was DSCAM plus COL6A2.

Figure 2. DSCAM and COL6A2 double transgenic mice exhibit cardiac hypertrophy.

a) Immunostains of COL6A2 and DSCAM (red) and nuclei (blue) in wild type (WT) and COL6A2 and DSCAM double transgenic mice (Double Tg) in adjacent consecutive sections of 3 month old adult hearts (Bar  =  11 µm). COL6A2 staining is predominantly extracellular (arrows), while DSCAM staining consists of two components; a general plasma membrane surface expression, most notable between adjacent cells (arrows), that would overlap with COL6A2 expression, and intracellular perinuclear staining (arrowhead), which may represent ER-Golgi to plasma membrane transport intermediates of DSCAM. b) Immunoblot analysis of DSCAM and COL6A2 expression in wild-type versus double transgenic mice. GAPDH served as a loading control. c) Increased heart weight in dissected hearts of DSCAM and COL6A2 double transgenic mice (N = 7) compared to their wild-type littermates (N = 5) (± SEM, * t-test (2 tailed, unequal variance) P<0.05). d) Hypertrophy detected by micro-CT in DSCAM and COL6A2 double transgenic hearts. e) Measurements of heart wall thickness (in mm ± SEM) of right ventricle (RV) left Ventricle (LV) and interventricular septum (IVS) derived from Micro-CT virtual sections of double transgenic (N = 8) and wild type (N = 2) hearts (* t-test (2 tailed, unequal variance) P<0.05). Bars in panel d indicate sites of measurement. f) 3-D reconstructions of WT versus double transgenic mouse hearts obtained from micro-CT analysis showing extensive hypertrophy in a frontal section of an adult double transgenic mouse heart. Left ventricle (LV) and right ventricle (RV) are indicated. g) Double transgenic heart myocytes exhibit increased cell size (dotted outlines indicate the borders of individual cells). IVS cardiomyocytes from heart sections stained with fluorescent wheat germ agglutinin. h) Quantification of cell size in IVS from stained heart sections (N = 20, * t-test - 2 tailed, unequal variance, P<0.0001).

Figure 3. Atrial septal defects in DSCAM and COL6A2 double transgenic mice.

a) Atrial Septal Defects (ASD) in a digitally reconstructed double transgenic heart based on Micro-CT imaging. High magnification insets show atrial septal region indicated by dotted lines in the lower magnification full views. Red arrows indicate a hole between left and right atria that also appear on adjacent reconstructed sections indicating that an open passage exists between the two atrial chambers. Atrial septum (AS), right atrium (RA), interventricular septum (IVS), Foramen ovale (FO). b) An ASD in an experimentally dissected double transgenic mouse. A frank hole between the left and right atria allowed the unobstructed passage of a course bristle. c) Digital Subtraction Angiography (DSA) analysis shows shunting of the radio opaque dye from the left atrium to the right atrium. DSA images from double transgenic mouse before (left) and during the detected shunting (right). Top panel-video capture, bottom-binary image after digital subtraction. Red arrows indicate the site of abnormal shunting of the dye from the left atrium into the right atrium. d) Saline contrast echocardiography. Abnormal shunting is detected in double transgenic mice as bubbles in the LV within 1 beat. Panels depict echocardiograms of the cardiac chambers in the axial plane of the heart, both before and after injection of saline. Blue arrows indicate time of injection, red arrows indicate the time bubbles are detected in LV, and yellow arrows indicate bubbles. Each panel consists of 10 frames merged for visualization purposes. e) Percent of wild-type and double transgenic animals exhibiting shunting in digital subtraction angiography and saline contrast echocardiography.

Figure 4. DSCAM and COL6A2 enhance cell adhesion and induce transcriptional changes.

a) Cell adhesion assay in a cardiomyocyte cell line. A stable line of H9C2 cardiomyocyte cells expressing DSCAM (H9C2-DSCAM) or parental H9C2 cells were grown on BSA or COL6 coated plates. At the indicated time points, plates were gently washed and the number of cells retained on the plates was determined shown as percent of cells adhering to plates. b) The top gene ontology KEGG pathway terms of differentially expressed genes identified by RNA-seq of H9C2 and H9C2-DSCAM cell lines grown on COL6 coated plates. Threshold shown p<0.05. c) Enriched heart related gene ontology terms in H9C2 vs. H9C2-DSCAM differentially expressed mRNAs using Ingenuity knowledge pathway database. Threshold shown p<0.05. d) qRT-PCR of select genes in wild type and double transgenic e14.5 hearts. Results shown as average +/− SEM (N = 3). Unpaired two-tailed t-test analysis p<0.05 (*).