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The Complex Genetics of Hypoplastic Left Heart Syndrome

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The Complex Genetics of Hypoplastic Left Heart Syndrome LETTERS The complex genetics of hypoplastic left heart syndrome Xiaoqin Liu1, Hisato Yagi1 , Shazina Saeed1, Abha S Bais1, George C Gabriel1, Zhaohan Chen1, Kevin A Peterson2, You Li1, Molly C Schwartz1, William T Reynolds1, Manush Saydmohammed1, Brian Gibbs1, Yijen Wu1, William Devine1, Bishwanath Chatterjee1, Nikolai T Klena1, Dennis Kostka1, Karen L de Mesy Bentley3, Madhavi K Ganapathiraju4, Phillip Dexheimer5, Linda Leatherbury6, Omar Khalifa1, Anchit Bhagat1, Maliha Zahid1, William Pu7, Simon Watkins8, Paul Grossfeld9, Stephen A Murray2, George A Porter Jr10 , Michael Tsang1 , Lisa J Martin11 , D Woodrow Benson12, Bruce J Aronow5 & Cecilia W Lo1 Congenital heart disease (CHD) affects up to 1% of live births1. LV often appeared muscle bound with lumen obliteration (Fig. 1d). Although a genetic etiology is indicated by an increased Unlike other CHD phenotypes, HLHS exhibited nonmendelian seg- recurrence risk2,3, sporadic occurrence suggests that CHD regation, and only one HLHS mutant was recovered per line, except genetics is complex4. Here, we show that hypoplastic left heart for the Ohia line, which had two mutants (Supplementary Table 1). syndrome (HLHS), a severe CHD, is multigenic and genetically Exome sequencing of the eight HLHS lines recovered 330 coding heterogeneous. Using mouse forward genetics, we report what or splicing mutations, none of which were shared, thus indicating that is, to our knowledge, the first isolation of HLHS mutant mice HLHS is genetically heterogeneous (Supplementary Data 1). Mutations and identification of genes causing HLHS. Mutations from in five Notch-related genes (Rbpj, Ep300, Nap1l1, Ncoa1, and Tspan12), seven HLHS mouse lines showed multigenic enrichment in a pathway implicated in HLHS10,11, were identified. Network analysis12 ten human chromosome regions linked to HLHS5–7. Mutations identified seven interconnected functional modules (0.64 modularity; in Sap130 and Pcdha9, genes not previously associated with 2.7 scaled modularity) significantly different from random networks CHD, were validated by CRISPR–Cas9 genome editing in mice (0.59 ± 0.019 modularity (n = 1,000, mean ± s.d.); P value = 0.003) as being digenic causes of HLHS. We also identified one subject (Supplementary Fig. 3). These results suggested that HLHS may with HLHS with SAP130 and PCDHA13 mutations. Mouse involve disturbance of the cell cycle (KIFC3 and CDK1); muscle dif- and zebrafish modeling showed that Sap130 mediates left ferentiation (TRIM63); mitochondrial and Notch signaling (STAT3, ventricular hypoplasia, whereas Pcdha9 increases penetrance EGFR, and EP300); EGF, Wnt, and cadherin signaling (EGFR and of aortic valve abnormalities, both signature HLHS defects. CTNNB1); chromatin regulation (EP300); and cardiac hypertrophy These findings show that HLHS can arise genetically in a (TRIM63, EGFR, and SLC4A1) (Supplementary Data 2). Five HLHS combinatorial fashion, thus providing a new paradigm for the mouse lines had mutations in two or more genes in 10 of 14 human complex genetics of CHD. chromosome intervals associated (log of odds (lod) >2) with HLHS and LV outflow obstruction5–7 (Fig. 1e). Significant enrichment was Mouse chemical mutagenesis coupled with ultrasound phenotyping observed when two or more mouse HLHS genes were interrogated of 100,000 fetal mice has yielded a wide spectrum of CHD mutants8,9, across the intervals (odds ratio (OR), 322.5; confidence interval (CI), © 2017 Nature America, Inc., part of Springer Nature. All rights reserved. All rights part Nature. of Springer Inc., America, Nature © 2017 including the recovery of eight HLHS mutant lines (Supplementary 24.9–4,177.2; P = 5.6 × 10−10), thus supporting multigenic etiology Table 1 and Supplementary Figs. 1 and 2). Ultrasound detection of for HLHS (Fig. 1f). HLHS lines, relative to control (Fig. 1a), showed a narrowed aortic The Ohia line with two founder HLHS mutants contained five flow (Fig. 1b and Supplementary Video 1) and a small left ventricle homozygous mutations (Supplementary Data 1 and Supplementary (LV) with poor contractility (Fig. 1b and Supplementary Video 2). Fig. 1), only two of which are required for HLHS (Table 1): a splice- Hypoplasia of the LV, aorta, and mitral valve (MV)—essential features site mutation in Sap130, which encodes a Sin3A-associated protein of HLHS—were confirmed by necropsy and histopathology (Fig. 1c,d in the histone deacetylase complex13,14, and a missense mutation in and Supplementary Videos 3 and 4). As observed clinically, the small Pcdha9, which encodes the cell-adhesion protein protocadherinA9 1Department of Developmental Biology, University of Pittsburgh School of Medicine, Pittsburgh, Pennsylvania, USA. 2The Jackson Laboratory, Bar Harbor, Maine, USA. 3Pathology & Laboratory Medicine and the Electron Microscope Research Core, University of Rochester School of Medicine and Dentistry, Rochester, New York, USA. 4Department of Biomedical Informatics, University of Pittsburgh School of Medicine, Pittsburgh, Pennsylvania, USA. 5Department of Biomedical Informatics, Cincinnati Children’s Hospital Medical Center, Cincinnati, Ohio, USA. 6Division of Cardiology, Children’s National Medical Center, Washington, D.C., USA. 7Department of Cardiology, Boston Children’s Hospital, Boston, Massachusetts, USA. 8Department of Cell Biology, Center for Biological Imaging, University of Pittsburgh School of Medicine, Pittsburgh, Pennsylvania, USA. 9Division of Cardiology, University of San Diego School of Medicine, San Diego, California, USA. 10Department of Pediatrics, Division of Cardiology, University of Rochester School of Medicine and Dentistry, Rochester, New York, USA. 11Division of Human Genetics, Cincinnati Children’s Hospital Medical Center, Cincinnati, Ohio, USA. 12Pediatric Cardiology, Medical College of Wisconsin, Milwaukee, Wisconsin, USA. Correspondence should be addressed to C.W.L. ([email protected]). Received 24 January; accepted 24 April; published online 22 May 2017; doi:10.1038/ng.3870 1152 VOLUME 49 | NUMBER 7 | JULY 2017 NATURE GENETICS LETTERS R A Cr P0 E16.5 a Cr c e 2 5 10 +360.8 Cd Ao 10p11–q22.2 mm/s Cd P Ao TSPAN15_L1430 (Chr 10) Ao L PA 2p21–22.3 5q21.2 RV PA FER_L1432 (Chr 17) RV HNRNPLL_L1432 (Chr 17) BICC1_L1430 (Chr 10) DCP2_L1432 (Chr 18) CTNNA3_L3077 (Chr 10) PA LVRN_L1432 (Chr 18) 10q22.31 LV OTI3_L1709 (Chr 10) Control Control TSPAN14_L1430 (Chr 14) LV RV RV LV 11 12 12q13.13 14 –360.8 LV CCDC65_L1963 (Chr 15) mm/s FKBP11_L1963 (Chr 15) KRT6A_L1432 (Chr 15) R NERUOD4_L1709 (Chr 10) 14q24.2 R 11q22.1 KIAA0586_L1430 (Chr 12) b A PSEN1_L1709 (Chr 12) A P P FDX1_L464 (Chr 9) Ao Ao PA L RA L PA RV RV 16 18 20 20q11.21 CTNNBL1_L3077 (Chr 2) 16p13.13–q12.2 SLC12A5_L3077 (Chr 2) HLHS WFDC11_L1432 (Chr 2) HLHS PA LA LV LV Ao RV KAT8_L3077 (Chr 7) RV NOMO1_L1709 (Chr 7) LV LV 18q22.3 SERPINB7_L1709 (Chr 1) d f –10 Ao PA Ao PA FO OR P = 5.6 × 10 LA LA RA LA RA LV RA RV LV FO RV 1,000 LV LV RV LV RV RV LV Control (P0) Control RV Control (E14.5) Control 100 FO Ao PA Ao PA RV intervals linkage P = 0.07 FO RV LV 1 LV LV LV RV LV HLHS in mutations Mouse 0.25 RV LV RV Mutant (P0) RV Single Multiple Mutant (E14.5) mutation mutations Figure 1 Ohia HLHS phenotype and mouse HLHS mutations in human HLHS linkage intervals. (a–d) HLHS phenotype in Ohia mutants. (a,b) Ultrasound color-flow imaging of normal fetus (a), showing robust flow from the aorta (Ao) and pulmonary artery (PA). In the HLHS mutant (b), the aorta showed only a narrow flow stream, whereas the pulmonary artery showed robust flow. 2D imaging revealed hypoplastic LV (b), as compared with the normal-sized LV in the control (a). (c) Newborn (P0) or E16.5 hearts from wild-type and HLHS mutants. Hypoplastic aorta and LV are visible in the HLHS mutant. (d) Histopathology showing the cardiac anatomy of HLHS mutant and littermate control at birth (P0) and E14.5. Compared with controls, the HLHS mutant exhibited hypoplastic aorta and aortic valve atresia, hypertrophied LV with no lumen, and MV stenosis and patent foramen ovale (FO), arrowhead. LA, left atrium; RA, right atrium; A, anterior; P, posterior; L, left; R, right; Cr, cranial; Cd, caudal. Scale bars: a,b, 0.5 mm; c, 1 mm; d, 0.5 mm. (e) Mouse HLHS mutations in human chromosome regions linked to HLHS and left-ventricular outflow tract obstruction. Genes recovered from mouse HLHS lines found in human chromosome regions linked to HLHS and left-ventricular outflow tract obstructions are shown5–7. The mouse chromosome location of each gene is indicated in parentheses. Mouse mutant lines are identified with L and a numerical identifier. Chr, chromosome. (f) Enrichment of mouse HLHS mutations recovered from seven mutant lines in human chromosome linkage intervals. Genes with mouse HLHS mutations in groups of two or more showed significant enrichment in the HLHS linkage intervals previously identified5–7 (Pearson chi-squared test). This result was not observed for genes analyzed individually, thus indicating that HLHS involves multigenic interactions. (Supplementary Fig. 4a,b). Both genes are expressed in the embry- normal-sized LV with isolated aortic hypoplasia and aortic steno- onic heart (Supplementary Fig. 4e,h–l). Ohia showed transmis- sis and bicuspid aortic valves (BAV), phenotypes linked to HLHS5 sion of HLHS and related CHD phenotypes, including isolated (Fig. 2a–c and Table 1). These findings were accompanied by cardiac hypoplastic LV or aorta, and outflow malalignment defects. Some hypertrophy, which was probably secondary to the aortic stenosis mutants had a double-outlet right ventricle (RV) with hypoplastic (Supplementary Videos 5–7). This same phenotype was observed m/m m/+ © 2017 Nature America, Inc., part of Springer Nature. All rights reserved. All rights part Nature. of Springer Inc., America, Nature © 2017 LV and normal aorta, or hypoplastic aorta and normal LV (Table 1 with increased penetrance in Pcdha9 ; Sap130 mutants (25% and Supplementary Fig. 5a–c), thus indicating that, in contrast versus 11%; Table 1).
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