Blue EE , White JJ , Dush MK , Gordon WW , Wyatt BH , White P , Marvin CT , Helle E , Ojala T , Priest JR , Jenkins MM , Almli LM , Reefhuis J , Pangilinan F , Brody LC , McBride KL , Garg V , Shaw GM , Romitti PA , Nembhard WN , Browne ML , Werler MM , Kay DM , National Birth Defects Prevention Study , University of Washington Center for Mendelian Genomics , Mital S , Chong JX , Nascone-Yoder NM , Bamshad MJ .

UM1 HG006493 NHGRI NIH HHS , U24 HG008956 NHGRI NIH HHS , R01 HD095937 NICHD NIH HHS , CDP 13-003 HSRD VA, EP-D-18-001 EPA, U01 DD001304 NCBDD CDC HHS, U01 DD001227 NCBDD CDC HHS, U01 DD001032 NCBDD CDC HHS

             cardiac left ventricle morphogenesis

Xla Wt + capn2 MO (Fig. S3. B)

	Figure 1. Discovery association tests. (AD) Comparison of SKAT-O (A and B) and burden (C and D) approaches to gene-based rare variant aggregate association analysis. Manhattan (B and D) and QQ (A and C) plots demonstrate that CAPN2 is the most significantly associated gene tested, regardless of approach. A low median value for the SKAT-O QQ plot (A) suggests genomic deflation in this approach.
	Figure 2. CAPN2 variants identified in individuals with isolated hypoplastic left heart syndrome (iHLHS) Gene diagram of CAPN2 (GenBank: NM_001748.4) with locations of variants identified in individuals with iHLHS. Black dots represent variants present in the association subset of the cohort, blue dots represent variants identified in samples excluded from the association analysis due to lack of ancestry-matched controls.
	Figure 3. CRISPR-Cas9-mediated functional studies of CAPN2 variants in vivo Xenopus laevis embryos were injected with capn2 single guide RNA (sgRNA) alone (control; A), capn2 sgRNA plus Cas9 protein (to elicit capn2 indels and loss of Capn2 function; B), or capn2 sgRNA/Cas9 co-injected with WT capn2 mRNA (C), mutant capn2 mRNA engineered to contain the orthologous human CAPN2707C>T variant (707 mRNA; D), or mutant capn2 mRNA engineered to contain the orthologous human CAPN21112C>T variant (1112 mRNA; E). Injected F0 embryos were allowed to develop through cardiogenesis and immunostained for Troponin T (red) to highlight the outflow tract (oft) and ventricle (v) of the heart (top row); optical sectioning was used to visualize the internal ventricular chamber (arrows, bottom row). In contrast to controls (A), embryos with CRISPR-Cas9-mediated loss of function of Capn2 (B) develop a hypoplastic ventricle and reduced ventricular chamber. Normal phenotype (C) is restored by co-injecting exogenous WT capn2 mRNA, confirming specificity of the phenotype. Co-injection of 707 mRNA can also restore normal ventricle phenotypes (D), although the frequency of rescue was not statistically significant (F), indicating that the CAPN2707C>T variant may be a mildly hypomorphic allele. Co-injection of 1112 mRNA is unable to rescue the HLHS-like phenotype at all (E and F), indicating the CAPN21112C>T variant may represent a null allele with respect to function necessary for normal ventricular development. (F) Results were quantified from three independent trials (n = 1457 embryos per condition, per experiment); error bars indicate standard deviation. Significance of differences between the percentage of HLHS-like phenotypes in each condition is noted. Scale bars, 100 m.
	Supplemental Figure 1. PC1 and PC2 values for each sample included in analysis. Samples are colored by outcome (case/control) and demonstrate that the first two PCs are not partitioning samplesbased on outcome.
	Supplemental Figure 2. Representative mutations at the Xenopus capn2 locus elicited by injection of capn2 sgRNA and Cas9 protein. Sequencing of a subset of individual clones validates the presence of deleterious mutations in capn2 sgRNA injected embryos. The sgRNA target sequences are highlighted (sgRNA #1, top; sgRNA #2, bottom), PAM sequence are boxed, deletions (Δ ) are indicated by dashes, and insertions (+) are underlined. CRISPRinduced mutations include frameshift and premature termination (e.g., Δ 5, Δ 14, +1), nonsynonymous substitution (e.g., Δ 1/+1) or deletion of 1 or more amino acids (e.g., Δ 6, Δ 9).
	Supplemental Figure 3. Morpholino knockdown of Xenopus Capn2 elicits a hypoplastic ventricle phenotype. While control (un-injected) embryos (A, D) develop normal heart morphology, embryos injected with a Capn2 morpholino oligonucleotide (Capn2 MO) have hypoplastic ventricles (B, E). The phenotype is fully rescuable by co-injection of WT capn2 mRNA (C, F), confirming the specificity of the effect. G) Quantification of morpholino results from 3 different experiments (n= 8-24 embryos per condition, per experiment); error bars indicate standard deviation

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