Supplementary Material

Methods and Results

siRNA knock down of MCTP2 in cultured human aortic smooth muscle cells

Coarctation of the aorta is thought to arise from abnormal juxtaductal vascular smooth muscle proliferation/regulation in the ascending aorta. To further explore the functional significance of altered MCTP2, we used two siRNAs to knock down endogenous MCTP2 in cultured human aortic smooth muscle cells. Control siRNA was also used to differentiate the non-specific effects of siRNA transfection from the MCTP2 knock-down effects. Global gene expression profiles were then assessed in the treated cells. The efficacy of MCTP2 knockdown was assessed by real-time RT-PCR (48 h after transfection). All genes whose expression differed significantly at P < 0.01 with a fold change≥ 2 were identified. Three genes,ACTG2, ITGA5, andPDGFRA were selected and the differential expression was validated by quantitative real-time PCR (Fig. S4). We used Ingenuity Pathway Software to perform pathway enrichment analysis and network construction and analyzed the combined list of upregulated and downregulated genes to identify enriched biological functions and pathways. Within these functions, regulation of actin-based motility by Rho pathway were specifically enriched (ACTG2, BAIAP2,CDC42 and DIRAS3, p=7.09x10-e3) (Supplementary Material, Fig. S5, S6, S7, and S8). We then combined and analyzed the top 13 networks. The resulting network included 150 molecules, which consisted of both the upregulated and downregulated genes and their known interacting molecules. The combined network similarly identified interactions of genes that are involved in cellular movement (min p=1.2x10-5), development (min p=2.7x10-2), and growth and proliferation (min p=2.5x10e-4)among the top enriched biological functions (Supplementary Material, Fig. S5).

Supplementary Table S1. Summary of clinical features insubjects 1 and 2

Features / Subject 1 / Subject 2
Karyotype / 46,XY / 46,XX
Size of genomic imbalance / 2.2 Mb deletion / 2.2 Mb deletion
Genes involved / MCTP2 / MCTP2
Inherited / Maternal mosaic deletion / Maternal mosaic deletion
Gender / Male / Female
Age at evaluation / 3 yrs / 10 yrs
Cranium / FOC (-2SD), microbrachycephalic, frontal hair sweep and two posterior hair whorls, mild bitemporal narrowing and mild glabellar prominence / FOC (-2SD)
Microcephalic
Eyes, nose, ears / Full eyebrows, bilateral epicanthal folds, long eyelashes, broad nasal root and bridge, malar hypoplasia, thin and down-turned upper lip. Low set left ear / Prominent eyebrows with mild synophrys, broad nasal bridge
and root, slight downslant to the palpebral fissures, midfacial
hypoplasia, down-turned and thin upper lip. Maloccluded and
crowded dentition. Low set ears
Heart / CoA, congenital anomaly of the mitral valve, membranous subvalvar aortic stenosis, muscular VSD / CoA, VSD
Musculoskeletal / Height (<5%), narrow shoulders / Height (4%), bilateral fifth finger clinodactyly, tapering digits
Neuromuscular / Mild hypotonia / Decreased bulk of the musculature with mild hypotonia
Development / Mild delay / Moderate gross & fine motor delays. Significant language delays and social and adaptive delays.

Supplementary Table S2. Summary of rare missense variants in MCTP2 after analyzing 146 individuals with left-sided obstructive cardiac lesions

§ Missense variants seen each in 1/192 ethnically matched control samples

BAV (bicuspid aortic valve); AVS (aortic valve stenosis); MS (mitral stenosis); VSD (ventricular septal defect)

Supplementary Table S3. Effects of MCTP2 variants predicted by PolyPhen-2, SIFT and population based data

Variants / PolyPhen-2 / SIFT / SNPs3D / dbSNP id / 1000 Genomes, MAF if available / Exome variant server, MAF
R46H / Benign / Tolerated / Could not generate consistent profile / rs61735139 MAF 0.014* / Yes / No
R47H / Probably Damaging / Tolerated / Could not generate consistent profile / rs149133063 / No / 3/12987
A60T / Benign / Tolerated / Could not generate consistent profile / rs143256791 / 0.003 / 74/12916
G203D / Probably Damaging / Affects protein Function / Deleterious / No / <0.01 / No
Y235C / Probably Damaging / Tolerated / Non-Deleterious / rs191271656 / <0.01 / No
V475I / Benign / Tolerated / Non-Deleterious / No / <0.01 / No
A695V / Benign / Tolerated / Non-Deleterious / rs144209524 / 0.006 / 1/12989

*present in 1/36 individuals in the Coriell "apparently healthy collection" (dbSNP population 13321, AGI_ASP).

Figure S1. Breakpoint mapping of 15q terminal region in individuals with LVOT and septal lesions

Figure S2. (A)Mctp2 is expressed in the developing heart of embryonic day E9.5, E10.5, and E11.5 mouse embryos. Semi-quantitative RT-PCR for Mctp2 yields a 167 bp product; -RT, minus reverse transcriptase control. (B)XenopusMctp2 morphant embryos have significant decreases in Mctp2 transcript. RNA isolated from stage 25 control, indicated as C or Mctp2MO1 or Mctp2 MO2 embryos was used for RT-PCR with EF-1α control primers andMctp2 primers spanning the morpholino splice block site (Mctp2 Exon4-6).

Figure S3.Expression of Mctp2 in the atrioventricular canal. RT-PCR was performed using RNA obtained from whole embryos at E12.5 (lane 1) or endocardial cushion primitive valve tissue isolated from E14.5 embryos (lane 2). MyoD1 primers are myocardial specific and Tie2 primers are endocardial specific.

Figure S4. Conservation of MCTP2 amino acid residues and missense substitutions in patients 4-8. The grey background indicates the C2A domain; the conserved residues at amino acid positions, 203 and 235 are highlighted in yellow. The alteration of these residues in patients 7 and 8, affects the Ca2+ binding affinity of the C2A domain.

Figure S5.siRNA knock down of MCTP2 in cultured human aortic smooth muscle cells.

(A)Microarray analysis showing differential gene expression in wild-type and MCTP2 silenced human aortic smooth muscle cells, using Illumina Human HT-12v3 Expression BeadChips. All genes whose expression differed significantly at P < 0.01 with a fold change ≥ 2 in the two siRNAs are represented. (B) Validation with qRT-PCR. Three genes, ACTG2, ITGA5, and PDGFRA were selected to validate differential expression by quantitative real-time. Downregulation of MCTP2 is also shown, in keeping with the silenced human aortic smooth muscle cells. The qRT-PCR results confirm differential expression in the silenced cells, consistent with the microarray results.

Figure S6. Differentially expressed genes in significantly enriched pathways and gene networks related to cellular movement, growth and development. We evaluated the top differentially expressed genes (p<0.05) between the MCTP2silenced human aortic smooth muscle cells(two siRNA) and wild-type cells and performed pathway analysis using the Ingenuity Pathway Software (IPA). Global gene expression profiles were then assessed in the silenced cells.(A) We show significant enrichment (1.20x10e-5x<p<4.56x10e-2) of functional annotations involved in cellular movement.(B) Further, enrichment analyses of canonical functions show strong support for the Regulation of Actin Motility by Rho (p=7.09x10-e3) pathway.

(A)

(B)

Figure S7. Pathway of Actin-based motility by Rho signaling is enriched in the differential gene expression in the MCTP2 knockdown human aortic smooth muscle cells (ACTG2, BAIAP2,CDC42 and DIRAS3, p=7.09x10-e3), suggestingthat altered dosage of MCTP2 dysregulates Rho signaling and affects cytoskeleton reorganization, an important process in endocardial cushion formation.

Figure S8.Interaction network of the differentially expressed genes in the MCTP2 silenced human aortic smooth muscle cells. Known interactions among differentially expressed genes within the top enriched pathways reveal several genes that are involved in cellular migration, growthand development, in addition to cardiovascular-related phenotypes. Red molecules are upregulated, green molecules are downregulated and hollow molecules are not differentially expressed but are involved in the significantly enriched interaction network.