Members of the Competence Network for Congenital Heart Defects, Germany
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Members of the Competence Network for Congenital Heart Defects, Germany Hashim Abdul-Khaliq, Hans-Heiner Kramer, Felix Berger, Brigitte Stiller, Ulrike Bauer, Thomas Pickardt, Sabine Klaassen Family 49 Family 62 Family 226 Family 333 * * * * * * * * TOF AVS ASD ASD PAPVR TOF * * * * ASD PAPVD COA ASD * Family 346 Family 398 VSD * Family 489 * * VSD VSD * * VSD HCM * * * Family 545 BAV AVS ASD PDA Sv AS VSD Family 576 Family 645 Septal defect * * * COA AVS ASD BAV VSD * * * * BAV AVS AVS ASD BAV Family 702 * VSD VSD Family 732 * Family 720 AVSD * * * VSD * * Family 831 TOF TOF TOF Vring TGA VSD * * * * PDA VSD AVS * PDA * * * VSD BAV PFO ASD ASD2 PDA PDA COA Family 1117 Family 1121 * PVS ? * ASD VSD * * * TOF VSD ASD VSD Family 1319 Family 1151 * * * * 2 * ASD ASD * * ASD ASD * * EbA AVS AVS Family 1364 Family 1560 Family 1575 * * TOF VSD ASD VSD infPS TOF * * * VSD PVS PVS PVS VSD * VSD PVS Family 1710 ASD Family 1722 * ASD CHD * VSD VSD * COA TGA DCM ASD BAV SV, MVA COA PVS * DCM DCM ASD HLHS COA BAV Family 2077 Family 2261 * COA, BAV * * * PVS PVS PVS * * infPS infPS BAV ASD * * AVS ASD ASD BAV BAV Family 3500 Family 2558 Family 3315 * * * AVS VSD AVR * * HLHS COA PDA ASD BAV * PVA Family 3501 VSD Family 3503 ? ? * * * VSD HRHS EbA * 3 PVA PAA ASD PDA VSD Family 3505 Family 3540 * * BAV ASD AVS * * * HLHS HLHS TAPVR BAV COA Figure S1. Pedigrees of 32 Danish multiplex CHD families. Circles: females. Squares: males. White symbols: unaffected family members. Filled symbols: affected family members. Triangles: abortion. CHD was determined by manual inspection of patient files (black) or data from DNPR/interview with family members (grey). Exome sequenced individuals are marked with asterix. The following heart defects were identified; Aortic Valve Regurgitation (AVR), Aortic Valve Stenosis (AVS), Atrial Septal Defect (ASD), Atrioventricular Septal Defect (AVSD), Bicuspid Aortic Valve(BAV), Coarctation of the Aorta (COA), Dilated Cardiomyopathy (DCM), Ebstein’s Anomaly (EbA), Hypertrophic Cardiomyopathy (HCM), Hypoplastic Left Heart Syndrome (HLHS), Infundibular Pulmonary Stenosis (InfPS), Mitral Valve Atresia (MVA), Partial Anomalous Pulmonary Venous Return (PAPVR), Patent Ductus Arteriosus (PDA), Patent Foramen Ovale (PFO), Pulmonary Valve Atresia (PVA), Pulmonary Valve Stenosis (PVS), Single Ventricle (SV), Subvalvular Aortic Stenosis (SubAS), Tetralogy of Fallot (TOF), Total Anomalous Pulmonary Venous Return (TAPVR), Transposition of The Great Arteries (TGA), Vascular ring (Vring), Ventricular Septal Defect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igure S3. Homogeneity of the cohort. Population distances from each of the 90 individuals in the cohort to the 20 nearest neighbors (NN) were calculated. The distances compared to the the mean of the population in terms of standard deviations (Z-scores) is plotted on the x-axis. $% )DPLOLHV O ) ) O ) ) O ) ) ) ) ) ) ) ) ) ) ) ) ) ) ) ) ) ) ) ) &XPXODWLYHIUDFWLRQ ) ) ) ) ) ) 5HDGVDIWHUGXSOLFDWHUHPRYDO 0LOOLRQV ) ) O 'HSWK )LJXUH66HTXHQFLQJFRYHUDJHDQGQXPEHURIUHDGV$&XPXODWLYHGHSWKRIVHTXHQFLQJSHUIDPLO\%1XPEHURIUHDGVDIWHUUHPRYDORIGXSOLFDWHV :KROHH[RPHVHTXHQFLQJ 6\VWHPVDQDO\VLVRI&'*V '1$H[WUDFWLRQ &+'SDWLHQWV 33,QHWZRUNRI SURWHLQVZLWK :KROHH[RPHVHTXHQFLQJ REOLJDWHFDUULHUV VHHGSURWHLQV LQWHUDFWLRQV 0DSSLQJWRKJ VLJQL¿FDQWFOXVWHUV XQLTXHYDULDQWV 9DULDQWFDOOLQJ ,GHQWL¿FDWLRQRIFOXVWHUV ZLWKWUHVKROGSYDOXH 5HDGGHSWK! 9DULDQWTXDOLW\¿OWHULQJ 3KUHGFDOOTXDOLW\! ,GHQWL¿FDWLRQRIFOXVWHUV FOXVWHUVZLWKSURWHLQV 9DULDQWVSUHVHQWLQDOO ZLWK!&'*V HQFRGHGE\!&'*V DႇHFWHGIDPLO\PHPEHUV 0$)LQ* 3HUPXWDWLRQDQDO\VLV 7ZRFOXVWHUVHQULFKHGIRU&'*V )UHTXHQF\¿OWHULQJ 0$)LQ([$& SURWHLQV &'*V 0$)LQ'DQLVKH[RPHV N SURWHLQV &'*V 0$)LQ*HQRPH'HQPDUNFRKRUW *HQHRQWRORJ\ (QULFKPHQWIRUELRORJLFDO (ႇHFW¿OWHULQJ 5HPRYDORIQRQFRGLQJYDULDQWV SURFHVVHVLQYROYHGLQ DQG10'YDULDQWV HQULFKPHQWDQDO\VLV FDOFLXPVLJQDOLQJ 5DUHYDULDQWVVHJUHJDWLQJ XQLTXHUDUH ZLWK&+'LQIDPLOLHV YDULDQWVLQJHQHV )LJXUH62YHUYLHZRIWKHVHTXHQFLQJDQGGDWDDQDO\VLVSURFHVVHV Figure S6. Number of CDGs per family when all rare variants (left) or only high severity variants (right) were considered. High severity variants were defined as variants creating splicing defects, frameshifts, premature stop codons and missense variants predicted to be damaging by both Polyphen and SIFT. Figure S7. Overlap between CDGs in pairs of families. The fraction of overlap (FO) between CDGs are shown for each pair of families. For example, 11% of the CDGs identified in family 49 overlaps with CDGs identified in family 2558, which is indicated by a FO value of 0.11. Figure S8. Families with rare inherited variants in CDGs. Only genes mutated in two or more families are shown. Figure S9. Overlap between the 1,785 CDGs in our families and a curated list of 829 genes known to cause CHD in mice. (A) Overlap with all 1,785 CDGs. (B) Overlap with genes carrying mutations scored pathogenic by SIFT/Polyphen-2. Figure S10. Distribution of CHD genes across families. CHD genes identified in mouse models are shown here. The distribution of human CHD genes is shown in Supplemental Table 4. (A) All CHD genes affected by rare variants. (B) CHD genes affected by high severity variants. High severity variants were defined as variants creating splicing defects, frameshifts, premature stop codons and missense variants predicted to be damaging by both Polyphen and SIFT. 1XPEHURIJHQHVHWV &DOFLXPVLJQDOLQJJHQHVHW 1XPEHURISDWKRJHQLFPXWDWLRQVLQJHQHVHW )LJXUH6'LVWULEXWLRQRISDWKRJHQLFPXWDWLRQVLQUDQGRPJHQHVHWVUDQGRPJHQHVHWVRIJHQHVZHUH FUHDWHG7KHVL]HGLVWULEXWLRQRIHDFKVHWZDVVLPLODUWRWKHFDOFLXPVLJQDOLQJJHQHVHWWHVWHGLQWKHFDVHFRQWUROVWXG\ $'&< ES $'&< ES ,735 ES &$&1$6 ES &$&1$, ES &$&1$+ ES &$&1$' ES *5,$ ES 3/&% ES DQG1)$7 ES 3DWKRJHQLFPXWDWLRQV ZHUHGH¿QHGDVPXWDWLRQVZLWK0$)DQG03&VFRUH! A) B) N: N: adcy2a-MO+itpr1b-MO+plcb2-MO 41 adcy2a-MO2+itpr1b-MO2+plcb2-MO2-S 153 itpr1b-MO+plcb2-MO 31 plcb2-MO2-S 41 adcy2a-MO+plcb2-MO 49 itpr1b-MO2-S 103 adcy2a-MO+itpr1b-MO 33 adcy2a-MO2-S 86 plcb2-MO 26 plcb2-MO2 203 itpr1b-MO 45 itpr1b-MO2 85 adcy2a-MO 73 adcy2a-MO2 186 WT 28 std-MO 139 0% 20% 40% 60% 80% 100% WT 45)) normal laterality defects other morphological defects 0% 20% 40% 60% 80% 100% normal laterality defects other morphological defects C) mRNA mRNA plcb2 adcy2a -MO + mRNA adcy2a adcy2a -MO + mRNA plcb2 plcb2 )Lgure 61. A. Injection of sub-efficient doses of MOs. Note that only combinations of MOs result in significant numbers of heart defects among the embryos. B. Quantification of heart phenotypes in WT, controls and morphants injected with second set of MOs. S indicates that sub-efficient doses of MOs are injected. C. Rescue of adcy2a and plcb2 knockdown. Embryos injected with adcy2a or plcb2 RNA alone and in combinations with corresponding MOs, are displayed at 48 hpf. myl7 in situ hybridization marks the heart in these embryos. Figure S13. Efficiency of the splice blocking morpholinos (MOs) used against adcy2a, itpr1b and plcb2. Upper panel shows first set of MOs and lower panel shows second set of MOs. Aberrant splicing of adcy2a, itpr1b and plcb2 and decrease in corresponding WT mature mRNA is confirmed by RT-PCR analysis. Figure S14. Rescue of adcy2a and plcb2 knockdown. 48 hpf embryos injected with adcy2a or plcb2 RNA alone and in combinations with corresponding SP-MOs, are displayed. myl7 in situ hybridization marks the heart in these embryos. Table S1. Human orthologues to 829 genes known to be associated with CHD in mouse models. The list was compiled from data in the Mouse Genome Informatics database (http://www.informatics.jax.org/). ABCA5 CHD7 FGFRL1 INVS MTERF3 PPARG SPTBN1 ABCB8 CHMP5 FHOD3 IRAK1 MTERF4 PPARGC1A SRF ABI1 CHRD FKBP1A IRX3 MTMR12 PPARGC1B SRSF1 ABL1 CHST14 FKBP1B IRX5 MTO1 PPP1R13L SRSF10 ACACB CISD2 FLNA ISL1 MUS81 PPP2R3A SSBP2 ACADM CITED2 FLRT3 ITCH MYBPC3 PPP2R5C SSR1 ACADVL CLU FMOD ITGA5 MYCN PPP3CB STK39 ACE CLUAP1 FN1 ITGAV MYH10 PRDM1 SUFU ACKR3 CNTRL FOXA2 ITGB1 MYH6 PRDM16 T ACSL4 COL18A1 FOXC1 ITPA MYH7 PRDM6 TAB1 ACTC1 COL1A1 FOXC2 JAG1 MYL2 PRF1 TAL1 ACVR1 COL4A3BP FOXD3 JARID2 MYL7 PRICKLE1 TBC1D32 ACVR2A COMMD9 FOXG1 JMJD6 MYLK3 PRKAR1A TBX1 ACVR2B CRB2 FOXH1 JPH2 MYO10 PRKCI TBX18 ACVRL1 CREBBP FOXJ1 JUN MYO18B PROC TBX2 ADAM12 CRKL FOXM1 JUND MYOD1 PSEN1 TBX20 ADAM15 CSNK2A1 FOXO1 JUP MYOZ2 PSEN2 TBX3 ADAM17 CSRP2 FOXP1 KAT6A NACA PSKH1 TBX5 ADAM19 CSRP3 FOXP4 KCNH2 NCKAP1 PTCD2 TBX6 ADAM9 CST9