Selected segregation patterns of CNVs in LS-CHD pedigrees.

<p>See legend at the bottom of the figure for explanation of symbols. DNA numbers refer to Tables S1, S2, S3, S4, S5, S6, S7, S8, S9 for affected individuals in whom rare CNVs were identified. A.) In family 5, we identified a maternally inherited gain overlapping <i>PXDNL</i> and a paternally inherited insertion der(9)ins(X;9)(p11.22;q12) overlapping the Cornelia de Lange syndrome gene SMC1A in the severely affected propositus.(NB: Individual 2126 was not initially genotyped on the Affymetrix 6.0 panel and is therefore not described). B.) The severely affected propositus in family 54 showed three different rare CNVs: a paternally inherited gain overlapping SEMA5B, HSPBAP1, DIRC2 and PARP14, a paternally inherited loss of LIMS1, and a <i>de novo</i> partial duplication on chromosome1q21.1. C.) <i>De novo</i> occurrence and non-transmission of a large CNV gain (3817 kb) on chr4p16 overlapping the Ellis van Creveld region on chromosome 4. D.), E.) F.) Segregation of prioritized CNVs with disease in families 18, 21 and 39.</p

Karyotype der(9)ins(X;9)(p11.22;q12) in family 5.

<p>(a,b) FISH was performed on metaphase chromosomes obtained from peripheral blood with a labeled BAC clone that mapped within the detected copy gain (RP11-52N6, red) and a control probe mapped to the Xp/Yp pseudoautosomal region of the sex chromosomes (DXYS129 & DXYS153, green). Green dots show the control probe hybridized to the p arm of chromosomes X and Y. Red dots show the RP11-52N6 BAC clone hybridized on chromosome X (white arrow heads) and in the heterochromatin of chromosome 9 (white arrows). A star shows the normal chromosome 9. These results show that the copy gain is due to a der(9)ins(X;9)(p11.22;q12) in both the father (a) and his son (b). (c). Chromosomal region of the insertion (X;9)(p11.22;q12) in the father and the son of family 5. Four RefSeq genes are identified within this <i>region IQSEC2, RIBC2, HSD17B10</i> and the Cornelia de Lange gene <i>SMC1A</i>. One larger and one smaller CNV have been detected in the DGV database in this region.</p

Overview over lesions.

<p>Distribution of isolated and combined LS-CHD phenotypes. Percents are relative to the total of 174 individuals with cardiovascular malformation.</p>*<p>This number includes one case with hypoplastic left heart syndrome.</p

Summary overview of the workflow for CNV detection.

<p>Flowchart of sample analysis from recruitment to prioritization.</p

dup(4)(p16.1) in family 43.

<p>FISH was performed on metaphase chromosomes and nuclei obtained from peripheral blood with a labeled BAC clone mapped within the detected copy gain (RP11-89K12, green) and a control probe mapped to 4p14 (RP11-332F10, red). (a) Two series of adjacent green dots show the extra copy of the duplicated segment on chromosome 4. (b) The nucleus view with the three green dots showing three copies of the region overlapping the Ellis van Creveld genes on chromosome 4 (c) Log 2 ratio for the large gain in Family 43 on chromosome 4. In general, dots are scattered around 0 along the x-axis for, whereas the identified gain leads to a clear upward shift (d) Heatmap of the identified gain on chromosome 4, each line refers to one individual. An orange row indicates two copies of the region whereas an extra copy leads to a gain in the intensity (yellow line for the individual in family 43).</p

Identified candidate genes.

<p>Compilation of the 25 LS-CHD candidate genes fulfilling all selection criteria. From left to right: gene name; ENDEAVOUR <i>in silico</i> prioritization; fold overexpression in SAGE experiments outflow tract versus atria/ventricles; <i>in situ</i> hybridization in mouse hearts at embryonic day E10.5; transmission pattern (individual IDs); genomic location.</p

mRNA expression profile of <i>CTHRC1</i> and <i>MFAP4</i> in embryonic mouse heart.

<p>(a, b). <i>In situ</i> hybridizations for <i>MFAP4</i> of a sagittal section of a wild-type stage E 14.5 mouse heart (c, d) <i>In situ</i> hybridizations for <i>CTHRC1</i> of a sagittal section of a wild-type stage E 14.5 mouse heart. Both assays show a strong expression in the pulmonary valve (arrows) and aortic/mitral valve (arrowheads). Unlike <i>CTHRC1</i> which is more restricted to the valves and only weakly expressed in the endothelium of the aorta, <i>MFAP4</i> shows a strong expression in the pulmonary artery and ascending aorta (asterisks). Pictures are taken from Eurexpress (<a href="http://www.eurexpress.org" target="_blank">www.eurexpress.org</a>).</p