A New ISL1 Loss-Of-Function Mutation Predisposes to Congenital Double

CLINICAL STUDY

ANewISL1 Loss-of-Function Mutation Predisposes to Congenital Double Outlet Right Ventricle

Zhi Wang,1＊ MD, Hao-Ming Song,2＊ MD, Fei Wang,3＊ MD, Cui-Mei Zhao,2 MD, Ri-Tai Huang,4 MD, Song Xue,4 MD, Ruo-Gu Li,5 MD, Xing-Biao Qiu,5 MD, Ying-Jia Xu,6 MD, Xing-Yuan Liu,1 MD and Yi-Qing Yang,6,7,8 MD

Summary Occurring in about 1% of all live births, congenital heart defects (CHDs) represent the most frequent type of developmental abnormality and account for remarkably increased infant morbidity and mortality. Aggregating studies demonstrate that genetic components have a key role in the occurrence of CHDs. Nevertheless, due to pronounced genetic heterogeneity, the genetic causes of CHDs remain unclear in most patients. In this research, 114 unrelated patients affected with CHDs and 218 unrelated individuals without CHDs served as controls were recruited. The coding regions and splicing donors/acceptors of the ISL1 gene, which codes for a transcription factor required for proper cardiovascular development, were screened for mutations by sequencing in all study participants. The functional characteristics of an identified ISL1 mutation were delineated with a dual-luciferase reporter assay system. As a result, a new heterozygous ISL1 mutation, NM_002202.2: c.225C>G; p.(Tyr75*), was discovered in an index patient with double outlet right ventricle and ventricular septal defect. Analysis of the proband’s family unveiled that the mutation co-segregated with the CHD phenotype. The nonsense mutation was absent in the 436 control chromosomes. Biological analysis showed that the mutant ISL1 protein had no transcriptional activity. Furthermore, the mutation nullified the synergistic activation between ISL1 and TBX20, another CHD-associated transcription factor. This research for the first time links an ISL1 loss-of-function muta- tion to double outlet right ventricle in humans, which adds insight to the molecular pathogenesis underpinning CHDs, suggesting potential implications for timely personalized management of CHD patients. (Int Heart J Advance Publication) Key words: Congenital heart defect, Cardiovascular development, Molecular genetics, Transcription factor, Transgene, Reporter gene assay

ongenital heart defects (CHDs), which are attrib- artery anomaly, patent ductus arteriosus, Ebstein’s anom- utable to abnormal development of the heart and aly, tetralogy of Fallot, double outlet right ventricle C cardiothoracic great vessels during embryogene- (DORV), aortic stenosis, transposition of the great arteries, sis, represent the most frequent kind of birth anomalies in truncus arteriosus, interrupted aortic arch, coarctation of humans, with an estimated prevalence of 1% in all live the aorta, pulmonary stenosis, endocardial cushion defect, births, and account for about one-third of all major con- hypoplastic left ventricle, and anomalous pulmonary ve- genital abnormalities.1) Every year, approximately 1.35 nous connection.1,2-8) Although minor CHDs may resolve million neonates are born with various CHDs in the spontaneously,2) severe CHDs require timely intervention world.2) Based on final anatomic and physiological pheno- in the first year of life and otherwise may give rise to re- types, CHDs have been classified into at least 25 distinc- duced exercise capacity and poor health-related quality of tive types, such as ventricular septal defect (VSD), life,9-11) neurodevelopmental anomaly or brain injury,12-14) double-orifice mitral valve, atrial septal defect, coronary pulmonary arterial hypertension,15-19) hemorrhagic or

From the 1Department of Pediatrics, Tongji Hospital, Tongji University School of Medicine, Shanghai, China, 2Department of Cardiology, Tongji Hospital, Tongji University School of Medicine, Shanghai, China, 3Department of Neurosurgery, Tongji Hospital, Tongji University School of Medicine, Shanghai, China, 4Department of Cardiovascular Surgery, Renji Hospital, School of Medicine, Shanghai Jiao Tong University, Shanghai, China, 5Department of Cardiology, Shanghai Chest Hospital, Shanghai Jiao Tong University, Shanghai, China, 6Department of Cardiology, The Fifth People’s Hospital of Shanghai, Fudan University, Shanghai, China, 7Department of Cardiovascular Research Laboratory, The Fifth People’s Hospital of Shanghai, Fudan University, Shanghai, China and 8Department of Central Laboratory, The Fifth People’s Hospital of Shanghai, Fudan University, Shanghai, China. *These authors contributed equally to this work. This work was supported by the grants from the National Natural Science Foundation of China (81470372, 81400244, and 81641014), and the Natural Sci- ence Foundation of Shanghai, China (16ZR1432500). Address for correspondence: Xing-Yuan Liu, MD, Department of Pediatrics, Tongji Hospital, Tongji University School of Medicine, 389 Xincun Road, Shanghai 200065, China. E-mail: [email protected] or Yi-Qing Yang, MD, Department of Cardiovascular Research Laboratory, The Fifth People’s Hospital of Shanghai, Fudan University, Shanghai 200240, China. E-mail: [email protected] Received for publication December 4, 2018. Revised and accepted April 16, 2019. Released in advance online on J-STAGE September 4, 2019. doi: 10.1536/ihj.18-685 All rights reserved by the International Heart Journal Association. 1 IntHeartJ 2 WANG, ET AL Advance Publication thromboembolic stroke,20-22) dementia,23) infective endo- Methods carditis,18-30) myocardial infarction,31,32) myocardial fibro- sis,33-35) cardiac dysfunction or chronic congestive heart Study subjects: Participants in this research include 114 failure,36-41) cardiac dysrhythmias,42-49) and sudden cardiac unrelated patients with CHDs (62 males and 52 females, death.50-53) Therefore, CHDs remain as the leading cause of with an average age of 3 years, ranging from 0-9 years of infant death from birth defects, with nearly 24% of new- age) and 218 unrelated healthy subjects without CHDs borns who died of birth deformities having CHDs.2) Al- used as controls (118 males and 100 females, with a mean though enormous advance in medical and surgical treat- age of 3 years, varying from 1-9 years of age). They were ments of CHDs has allowed over 90% of neonates with enrolled from the Chinese Han population. In addition, CHDs to survive into adulthood, it results in an ever- the available relatives of the index patient harboring a dis- increasing population of adult patients who are living with covered ISL1 mutation were also included. All study par- CHDs, and now, the adults with CHDs outnumber the ticipants experienced comprehensive clinical investigation, children with CHDs.1,54) In addition, the morbidity and including review of medical history, such as cardiac cathe- mortality in adult patients affected with CHDs are much terization and/or surgery for CHDs, detailed physical ex- higher than those in the general population.55-58) In spite of amination, two-dimensional echocardiogram with color important clinical significance, the causes of CHDs re- Doppler flow, and standard 12-lead electrocardiogram. In- main largely obscure. dex patients with known chromosomal abnormalities or It has been generally understood that heart develop- syndromic CHDs, such as 22q11.2 microdeletion, Turner ment is a complex biological process, and both inherited syndrome, Down syndrome, Noonan syndrome, Marfan and environmental pathogenic factors may interfere with syndrome, and Holt-Oram syndrome, were excluded from the process, resulting in CHDs.59-61) The well-established the present investigation. This study was carried out in ac- environmental risk factors for CHDs include maternal cordance with the ethical standards of the Declaration of conditions, such as viral infection and autoimmune disor- Helsinki 1964 and its later amendments and was approved der, and maternal exposures to toxic chemicals, as well as by the Research Ethics Committees of Tongji Hospital, ionizing radiation during the first trimester of pregnancy.59) Tongji University, Shanghai, China (ethical approval num- However, increasing investigations highlight the major ber: LL(H)-09-07; date of approval: July 27, 2009). In- contribution of genetic determinants to CHDs, especially formed consent was obtained from the legal guardians of to familial CHDs, which are transmitted in an autosomal the CHD cases and the control individuals prior to the in- dominant, autosomal recessive, or X-linked pattern in the vestigation. family.59-61) Irrespective of chromosomal abnormalities, in- Mutational screening of ISL1: About 2 μL of venous cluding trisomy of chromosome 21, trisomy of chromo- blood sample was collected from each study participant. some 13, trisomy of chromosome 18, and deletion of Genomic DNAs from blood leukocytes were isolated us- chromosome 22q11, as well as other copy number vari- ing the QIAamp DNA Mini Kit (Qiagen, Hilden, Ger- ations,59-61) an increasing number of mutations in over 60 many) following the manufacturer’s descriptions. The genes, encompassing those coding for cardiac transcrip- primers for amplification of the coding exons and exon- tion factors, myocardial structural proteins, signaling intron boundaries as well as partial 3’- and 5’-untranslated molecules, and chromatin modifiers, has been implicated regions of ISL1 by polymerase chain reaction (PCR) were in the pathogenesis of CHDs in humans.59-109) Among these designed as described previously.116) All coding exons and CHD-causing genes, the majority encode cardiac core flanking introns of ISL1 were amplified by PCR with Hot- transcription factors, encompassing zinc finger transcrip- Star Taq DNA Polymerase (Qiagen) on a Veriti Thermal tion factors GATA4-6; homeobox-containing transcription Cycler (Life Technologies, Carlsbad, CA, USA). The puri- factors NKX2.5, HAND1, and HAND2; and T-box tran- fied amplicons were sequenced with the BigDyeⓇ Termi- scription factors TBX1, TBX5, and TBX20.59-109) Neverthe- nator v3.1 Cycle Sequencing Kit (Life Technologies) un- less, CHDs are of substantial genetic heterogeneity, and der an ABI PRISM 3130 XL DNA Analyzer (Life Tech- the genetic defects underpinning CHDs in the vast major- nologies). Besides, the Single-Nucleotide Polymorphism ity of cases are still to be identified. (SNP) database (https://www.ncbi.nlm.nih.gov/snp/), As an important member of the homeobox family of Exome Aggregation Consortium (ExAC) database (http:// transcription factors, ISL1 is amply expressed in the em- exac.broadinstitute.org/), Human Gene Mutation Database bryonic heart and is key to normal cardiovascular morpho- (HGMD; http://www.hgmd.cf.ac.uk/ac/all.php), 1000 genesis.110-113) In mice, targeted deletion of the Isl1 gene Genomes Project (1000GP) database (http://www.interna- led to embryonic demise due to severe developmental tionalgenome.org/), Exome Variant Server (EVS) database malformations of the heart, encompassing loss of the right (http://evs.gs.washington.edu/EVS), and Genome Aggrega- ventricle, outflow tract, and much of the atria.110) In hu- tion Database (gnomAD; http://gnomad.broadinstitute.org/) mans, multiple variants in ISL1 have been reported to were retrieved to validate the novelty of an identified ISL1 confer an enhanced vulnerability to CHDs in diverse co- variation. horts of patients.114-116) These results make it justifiable to Plasmid constructs and site-targeted mutagenesis: The scan ISL1 for mutations underlying CHDs in another co- wild-type ISL1-pcDNA3.1 and TBX20-pcDNA3.1 expres- hort of patients. sion plasmids and the reporter plasmid MEF2C-luciferase (MEF2C-luc), which expresses Firefly luciferase, were constructed as previously described.116) The mutation iden- tified in CHD patients was introduced into the wild-type IntHeartJ Advance Publication ISL1 MUTATION IN DORV 3

ISL1-pcDNA3.1 plasmid by site-directed mutagenesis echocardiograms displayed normal cardiac images with no with the QuikChangeⓇ XL Site-Directed Mutagenesis Kit structural cardiac defects. The baseline clinical characteris- (Agilent Technologies, Santa Clara, CA, USA), using a tics of the CHD patients are shown in Table I. complementary pair of primers (forward primer: 5’-TTAT Discovery of a new ISL1 mutation: Sequence analysis ATCAGGTTGTAGGGGATCAAATGCGCC-3 ’ ; reverse of the whole coding regions and splicing junction sites of primer: 5’-GGCGCATTTGATCCCCTACAACCTGATATA the ISL1 gene was completed in 114 unrelated patients af- A-3’) according to the manufacturer’s protocol, and was fected with CHDs, and a mutation, NM_002202.2: c.225C confirmed by sequencing. To construct a control expres- >G; p.(Tyr75*), was discovered in an index patient aged 1 sion plasmid, the most common polymorphism (rs year. Specifically, a substitution of guanine for cytosine at 2303751) of c.504A>G (p.Pro168Pro), with an allele gene the third nucleotide of codon 75 (c.225C>G), predicting frequency of 0.3739 in the general population reported in the change of the codon encoding tyrosine at amino acid the ExAC database (http://exac.broadinstitute.org/variant/5- position 75 into a premature termination codon, p. 50685505-A-G), was introduced into the wild-type ISL1- (Tyr75*), was identified in a girl with DORV and VSD, pcDNA3.1. who had a positive family history of CHD. The sequence Cell culture, transient transfection, and luciferase chromatograms showing the heterozygous ISL1 mutation analysis: Chinese hamster ovary (CHO) cells and 10T1/2 of c.225C>G and its wild-type sequence are exhibited in cells were cultivated in Dulbecco’s Modified Eagle Me- Figure 1A. The schematic drawings of the Tyr75*-mutant dium added with 10% fetal bovine serum (Sigma-Aldrich, and wild-type ISL1 proteins denoting the main structural St. Louis, MO, USA), as well as 100 U/mL penicillin domains and location of the mutation discovered in the (Sigma-Aldrich) and 100 μg/mL streptomycin (Sigma- present research are displayed in Figure 1B. The pedigree Aldrich), in an incubator with an atmosphere of 5% CO2 structure of the proband’s family is exhibited in Figure 1 at 37°C. Cells were grown in 12-well plates at a density C. Sequencing analysis of the ISL1 gene in the proband’s of 1 × 105 cells per well. After 48 hours, cellular transfec- relatives available showed that the mutation was present in tion was carried out with EffecteneⓇ transfection reagent all the affected relatives (II-3, II-6, III-2) but absent in un- (Qiagen) following the manufacturer’s instructions. Cellu- affected relatives (I-2, II-2, II-4). Genetic analysis of the lar transfections with various plasmids were performed as proband’s pedigree revealed that the truncating mutation previously described.116) The pGL4.75 vector (Promega, co-segregated with the CHD phenotype, which was trans- Madison, WI, USA), which expresses a Renilla luciferase, mitted in an autosomal dominant pattern in the family was co-transfected into the cells as an internal control to with complete penetrance. The phenotypic characteristics normalize transfection efficiency. As two negative con- of the proband’s affected family members are given in Ta- trols, the empty pcDNA3.1 plasmid with no ISL1 expres- ble II. The nonsense mutation was neither observed in the sion and the pcDNA3.1 plasmid expressing Pro168Pro- 218 control persons nor found in the SNP, ExAC, HGMD, ISL1 were used. Cells were cultured at 37°C and col- 1000GP, EVS, and gnomAD databases (consulted again lected 48 hours after transfection. Luciferase activity of on December 3, 2018). the cell lysates was measured in a GloMaxⓇ 96 Mi- The mutant ISL1 protein lost transactivation of the croplate Luminometer (Promega), with the dual-luciferase MEF2C promoter: Previous experiments in vivo and in reporter assay system (Promega) according to the proto- vitro have demonstrated that ISL1 transcriptionally acti- col. The results were expressed as the ratios of the activi- vates the promoter of MEF2C.116-118) As displayed in Fig- ties of Firefly luciferase to Renilla luciferase. For each ex- ure 2, 1.0 μg of wild-type ISL1-pcDNA3.1 plasmid (ISL pression plasmid, three independent experiments were 1) and 1.0 μg of Tyr75*-mutant ISL1-pcDNA3.1 plasmid done in triplicate, and the MEF2C promoter activity was (Tyr75*) transactivated the MEF2C promoter by ～13- given as mean ± standard deviation. folds and ～1-fold, respectively. In addition, when 0.5 μg Statistical analysis: The statistical analyses were made of wild-type ISL1-pcDNA3.1 (ISL1) was utilized in com- using the SPSS for Windows statistical software package bination with 0.5 μg of empty pcDNA3.1 or 0.5 μg of (SPSS, Chicago, IL, USA). Continuous variables were Tyr75*-mutant ISL1-pcDNA3.1 (Tyr75*), the resultant compared between two groups using Student’s unpaired t- transactivation of the MEF2C promoter was the same of test, whereas categorical variables were compared between ～7-folds. These results indicate that the Tyr75*-mutant two groups using Pearson’s χ2 test or Fisher’s exact test, ISL1 has no transactivation on the promoter of MEF2C. as indicated. A two-tailed P-value of < 0.05 was consid- Synergistic transactivation between ISL1 and TBX20 ered significantly different. disrupted by the mutation: As given in Figure 3, the wild-type and Tyr75*-mutant ISL1 transactivated the MEF 2C promoter by 6-folds and 1-fold, respectively. Results ～ ～ When the wild-type TBX20-pcDNA3.1 plasmid (which Clinical features of the study subjects: In this research, alone transcriptionally activated the MEF2C promoter by a cohort of 114 unrelated CHD patients was clinically ～10-folds) was co-transfected, the wild-type and Tyr75*- evaluated in contrast to a total of 218 unrelated control in- mutant ISL1 transactivated the MEF2C promoter by ～23- dividuals. Patients were matched with control individuals folds and ～9-folds, respectively. These data suggest that for ethnicity, gender, and age. The cases had the Tyr75* mutation abrogates the synergistic transcrip- echocardiogram-documented CHDs, of whom roughly tional activity of ISL1 with TBX20. 32% had a positive family history of CHDs. The control subjects had a negative family history of CHDs, and their IntHeartJ 4 WANG, ET AL Advance Publication

Table I. Baseline Clinical and Demographic Characteristics of the Patients Affected with Congenital Heart Defects (n = 114)

Variable n or mean with SD % or range Gender Male 62 54 Female 52 46 Age (years) 3.1 ± 2.8 0-9 Positive family history of CHDs 37 32 Distribution of various types of CHDs Isolated CHDs 58 51 VSD 17 15 ASD 13 11 PDA 10 9 DORV 5 4 AS 5 4 PS 3 3 TGA 2 2 PTA 1 1 APVC 1 1 ECD 1 1 Complex CHDs 56 49 TOF 14 12 VSD + ASD 11 10 DORV + VSD 10 9 VSD + PDA 7 6 ASD + PDA 5 4 PTA + VSD 5 4 TGA + VSD 2 2 VSD + PS 1 1 TOF + ASD 1 1 Incidence of arrhythmias Atrioventricular block 8 7 Atrial fibrillation 5 4 Treatment Cardiac surgery 65 57 Catheter-based repair 32 28 Follow-up1715 SD indicates standard deviation; CHDs, congenital heart defects; VSD, ven- tricular septal defect; ASD, atrial septal defect; PDA, patent ductus arterio- sus; DORV, double outlet right ventricle; AS, aortic stenosis; PS, pulmonary stenosis; TGA, transposition of the great arteries; PTA, persistent truncus ar- teriosus; APVC, abnormal pulmonary venous connection; ECD, endocardial cushion defect; and TOF, tetralogy of Fallot.

the embryonic and postnatal hearts of rodents and humans Discussion and plays a fundamental role in normal cardiovascular In the present investigation, a new heterozygous mu- morphogenesis mainly by regulating the expression of key tation in the ISL1 gene, NM_002202.2: c.225C>G; p. target genes.111-113) The ISL1 protein possesses two struc- (Tyr75*), was detected in a family with DORV and VSD. tural domains, homeodomain and transcriptional activation The truncating mutation, which was absent in the 436 ref- domain, which are both pivotal to its function.116,118) The erential chromosomes, co-segregated with the CHD phe- evolutionarily conserved homeodomain, which consists of notype in the family with complete penetrance. Functional 60 amino acids from amino acids 181-240, functions to assays showed that the Tyr75*-mutant ISL1 protein lost bind the consensus DNA sequence in the promoters of transcriptional activity. Furthermore, the Tyr75* nullified target genes. The transcriptional activation domain, which the synergistic transcriptional activation between ISL1 and is immediately adjacent to the homeodomain comprising TBX20, another transcription factor that has been causally 109 evolutionarily conserved amino acids from amino ac- linked to CHD. Hence, it is very likely that genetically ids 241-349, is required for the transcriptional activation defective ISL1 predisposes to DORV as well as VSD in of target genes.116,118) In the current research, the mutation this family. discovered in a CHD family was anticipated to yield a The ISL1 gene is mapped on human chromosome 5q truncated protein with only 74 amino-terminal amino ac- 11.1, coding for a transcription factor protein comprising ids left, losing the DNA-binding and transactivation do- 349 amino acids. The ISL1 protein is highly expressed in mains, hence was expected to abrogate its transactivation IntHeartJ Advance Publication ISL1 MUTATION IN DORV 5

Figure 1. New ISL1 mutation responsible for congenital heart diseases. A: Sequence chromato- grams showing the heterozygous ISL1 mutation as well as its homozygous wild-type control. The arrow directs the heterozygous nucleotides of C/G in the index patient (mutant type) or the homozy- gous nucleotides of C/C in a control individual (wild-type). The rectangle marks the nucleotides comprising a codon of ISL1. B: Schematic drawings displaying the structural domains of the wild- type and Tyr75*-mutant ISL1 proteins as well as the location of the mutation identified in this re- search. COOH indicates carboxyl terminus; HD, homeodomain; NH2, amino terminus; and TAD, transcriptional activation domain. C: Pedigree structure of the family affected with congenital heart diseases. Family members are recognized by generations and numbers. Squares denote male family members; circles, female members; closed symbols, affected members; open symbols, unaffected members; a symbol with a diagonal, a dead member; an arrow, a proband; “ + ,” carriers of the het- erozygous mutation; “−,” non-carriers. of target genes, encompassing the MEF2C gene that has bryonic days 10.5-11, though heterozygous mice survived been previously reported to cause CHDs.77,98,119,120) Func- and showed no obvious anomaly.110) Histological analysis tional analyses showed that the Tyr75*-mutant ISL1 pro- of the homozygous knockout hearts between embryonic tein lost transactivation of the MEF2C promoter, alone or days 9.0 and 9.5 revealed severe developmental deformi- in synergy with TBX20, another transcriptional factor that ties of the heart, including a misshapen single ventricle is key to cardiogenesis and has been identified as an im- due to lack of the right ventricle, and absence of the out- portant gene responsible for CHDs.66) These observational flow tract as well as much of the atria.110) These observa- results provide strong evidence that haploinsufficiency tional results, along with cell lineage tracing experiments caused by ISL1 mutation is an alternative pathogenesis utilizing Cre mice expressing the Cre recombinase under underpinning CHDs. the Isl1 promoter, demonstrates that Isl1 marks undifferen- Association of genetically compromised ISL1 with tiated cardiac progenitors that contribute substantially to enhanced susceptibility to DORV may be ascribed at least the embryonic heart, comprising cells of the outflow tract, partially to abnormal cardiovascular development. In Isl1- right ventricle, both atria, and a subset of cells within the deletious mice, homozygous mutants for Isl1 exhibited left ventricle.121) growth retardation during embryogenesis and died at em- Previous studies demonstrated that several common IntHeartJ 6 WANG, ET AL Advance Publication

Table II. Phenotypic Features and ISL1 Mutation Status of the Family Mem- bers Affected with Congenital Heart Defects

Individuals Gender Age (years) Cardiac phenotypes ISL1 mutation Family 1Tyr75* I-1 M 47* DORV, VSD NA I-2 F 51 Normal −/− II-2 F 26 Normal −/− II-3 M 24 DORV, VSD +/− II-4 F 24 Normal −/− II-6 F 20 DORV, VSD +/− III-2 F 1 DORV, VSD +/− M indicates male; F, female; DORV, double outlet right ventricle; VSD, ventric- ular septal defect; NA, not available; −/−, wild-type homozygote; and +/−, het- erozygote. *Age at death.

Figure 2. No transcriptional activity of the mutant ISL1 protein on the MEF2C promoter. In cul- tured CHO cells, the Tyr75*-mutant ISL1 (Tyr75*) protein conferred no activation of the expres- sion of the MEF2C promoter-driven luciferase, when compared with its wild-type counterpart. For each expression plasmid, three independent cellular transfection experiments were carried out in triplicates. The results are shown by means and standard deviations. ## t = 10.1981, P = 0.0005, # t = 4.9657, P = 0.0077, in comparison with wild-type ISL1 (1.0 μg). polymorphisms in the ISL1 gene conferred an enhanced phisms (rs6869844, rs3762977, rs2115322, rs4865656, rs susceptibility to CHDs.114,115) Stevens and coworkers114) per- 6449600, rs1017, rs6449612, rs150104955, rs6867206) formed a case-control study of the 30 polymorphisms lo- and flanking ISL1 in 233 cases afflicted with CHDs and cated at the ISL1 locus in 300 pediatric cases affected 288 healthy control people and confirmed that only the with complex CHDs and 2,201 healthy control children polymorphism of rs1017 in ISL1 was significantly associ- and found that 8 polymorphisms (rs6449612, rs6449600, ated with increased risk for CHDs. Notably, Ma and part- rs4865656, rs2115322, IVS1+17C>T, rs6869844, rs1017, ners116) recently sequenced the coding exons and splicing rs6867206) in and near ISL1 were significantly associated boundaries of ISL1 in 210 unrelated patients with CHDs with enhanced vulnerability to CHDs. In order to inde- and detected a novel heterozygous mutation, c.409G>T or pendently verify the above-mentioned results, they made a p.E137X, in an index patient with congenital patent duc- replication research of these genetic polymorphisms in tus arteriosus and VSD. Analysis of the proband’s pedi- 1,044 new patients and 3,934 independent control indi- gree unveiled that the mutation, which was absent in 512 viduals and validated the significant association of these control chromosomes, co-segregated with the CHD pheno- polymorphisms with increased susceptibility to non- type. Functional analysis demonstrated that the E137X- syndromic CHDs.114) In order to ascertain whether the ISL mutant ISL1 protein failed to transactivate the promoter of 1 gene was associated with CHDs in the Chinese Han MEF2C. These data together with the findings from the population, Luo and colleagues115) analyzed 9 polymor- current study indicate that ISL1 exerts a pivotal role in the IntHeartJ Advance Publication ISL1 MUTATION IN DORV 7

Figure 3. Synergic transactivation between ISL1 and TBX20 nullified by the mutation. In the cultivated 10T1/2 cells co-transfected with the wild-type TBX20 expression plasmid, Tyr75*-mutant ISL1 (Tyr75*) protein conferred no transactivation of the firefly luciferase expression driven by the MEF2C promoter in synergy with TBX20, when compared with its wild-type counterpart. For each expression plasmid, three independent cellular transfection experiments were done in triplicates. The results are given as means with standard deviations. # t = 7.1534, P = 0.0020, in comparison with its wild-type counterpart. human heart development and congenital heart diseases. by a mediastinal hematoma after interrupted aortic arch sur- Notably, ISL1 loss-of-function mutations were previ- gery. Int Heart J 2017; 58: 629-32. ously linked to congenital patent ductus arteriosus, VSD, 7. Enomoto Y, Hashimoto G, Sahara N, et al. Congenital ab- 116,122) sence of left atrial appendage diagnosed by multimodality im- and dilated cardiomyopathy. Here, a new ISL1 loss- aging. Int Heart J 2018; 59: 439-42. of-function mutation was causally linked to DORV and 8. Liu S, Ren W, Ma C, Yang J. Congenital double-orifice mitral VSD. The various phenotypes linked to mutant ISL1 may valve in asymptomatic patients. Int Heart J 2018; 59: 213-5. be partially attributed to different genetic backgrounds, in- 9. Ko JM, White KS, Kovacs AH, et al. Physical activity-related complete penetrance, and genomic imprinting, which is drivers of perceived health status in adults with congenital associated with preferential expression of the paternal or heart disease. Am J Cardiol 2018; 122: 1437-42. maternal allele of a certain gene, underscoring the effect 10. Ko JM, Tecson KM, Rashida VA, et al. Clinical and psycho- 123) logical drivers of perceived health status in adults with con- of proband. genital heart disease. Am J Cardiol 2018; 121: 377-81. In conclusion, this study for the first time reveals that 11. Heusch A, Kahl HJ, Hensel KO, Calaminus G. Health-related ISL1 loss-of-function mutation predisposes to human quality of life in paediatric patients with congenital heart de- DORV, which helps to further understand the molecular fects: association with the type of heart defect and the surgi- mechanism underlying CHDs, implying potential implica- cal technique. Qual Life Res 2017; 26: 3111-7. tions for genetic counseling and personalized management 12. Morton PD, Ishibashi N, Jonas RA. Neurodevelopmental ab- normalities and congenital heart disease: insights into altered of the CHD patients. brain maturation. Circ Res 2017; 120: 960-77. 13. Peyvandi S, Chau V, Guo T, et al. Neonatal brain injury and timing of neurodevelopmental assessment in patients with congenital heart disease. J Am Coll Cardiol 2018; 71: 1986- Disclosures 96. Conflicts of interest: None. 14. Jørgensen DES, Tabor A, Rode L, et al. Longitudinal brain and body growth in fetuses with and without transposition of the great arteries. Circulation 2018; 138: 1368-70. References 15. van der Feen DE, Bartelds B, de Boer RA, Berger RMF. Pul- monary arterial hypertension in congenital heart disease: 1. Zaidi S, Brueckner M. Genetics and genomics of congenital translational opportunities to study the reversibility of pulmo- heart disease. Circ Res 2017; 120: 923-40. nary vascular disease. Eur Heart J 2017; 38: 2034-41. 2. Benjamin EJ, Virani SS, Callaway CW, et al. Heart disease 16. Schwartz SS, Madsen N, Laursen HB, Hirsch R, Olsen MS. and stroke statistics-2018 update: a report from the American Incidence and mortality of adults with pulmonary hyperten- Heart Association. Circulation 2018; 137: e67-492. sion and congenital heart disease. Am J Cardiol 2018; 121: 3. Numata G, Amiya E, Kojima T, et al. Cardiac resynchroniza- 1610-6. tion therapy in patients with Ebstein’s anomaly. Int Heart J 17. Cheng XL, Liu ZH, Gu Q, et al. Prognostic value of pulmo- 2017; 58: 816-9. nary artery compliance in patients with pulmonary arterial 4. Sakai C, Yamano T, Miki T, et al. Imaging of right-to-left hypertension associated with adult congenital heart disease. shunt in an adult patient with unroofed coronary sinus with Int Heart J 2017; 58: 731-8. persistent left superior vena cava. Int Heart J 2017; 58: 1008- 18. van Dissel AC, Mulder BJ, Bouma BJ. The changing land- 11. scape of pulmonary arterial hypertension in the adult with 5. Lee Y, Naruse Y, Tanaka K. Surgical treatment of coronary to congenital heart disease. J Clin Med 2017; 6: e40. pulmonary artery fistulas in adults. Int Heart J 2017; 58: 19. Kaemmerer H, Apitz C, Brockmeier K, et al. Pulmonary hy- 1012-6. pertension in adults with congenital heart disease: updated 6. Hua Q, Lin Z, Hu X, Zhao Q. Tracheal compression caused recommendations from the Cologne Consensus Conference IntHeartJ 8 WANG, ET AL Advance Publication

2018. Int J Cardiol 2018; 272S: 79-88. 40. Van De Bruaene A, Hickey EJ, Kovacs AH, et al. Phenotype, 20. Giang KW, Mandalenakis Z, Dellborg M, et al. Long-term management and predictors of outcome in a large cohort of risk of hemorrhagic stroke in young patients with congenital adult congenital heart disease patients with heart failure. Int J heart disease. Stroke 2018; 49: 1155-62. Cardiol 2018; 252: 80-7. 21. Pickard SS, Gauvreau K, Gurvitz M, et al. Stroke in adults 41. Soma K, Yao A, Saito A, et al. Regular treatment strategy with coarctation of the aorta: a national population-based with a large amount of carvedilol for heart failure improves study. J Am Heart Assoc 2018; 7: e009072. biventricular systolic failure in a patient with repaired tetral- 22. Lanz J, Brophy JM, Therrien J, Kaouache M, Guo L, Marelli ogy of Fallot. Int Heart J 2018; 59: 1169-73. AJ. Stroke in adults with congenital heart disease: incidence, 42. Niu MC, Dickerson HA, Moore JA, et al. Heterotaxy syn- cumulative risk, and predictors. Circulation 2015; 132: 2385- drome and associated arrhythmias in pediatric patients. Heart 94. Rhythm 2018; 15: 548-54. 23. Bagge CN, Henderson VW, Laursen HB, Adelborg K, Olsen 43. Drakopoulou M, Nashat H, Kempny A, et al. Arrhythmias in M, Madsen NL. Risk of dementia in adults with congenital adult patients with congenital heart disease and pulmonary ar- heart disease: population-based cohort study. Circulation terial hypertension. Heart 2018; 104: 1963-9. 2018; 137: 1912-20. 44. McLeod CJ. Acute arrhythmias in adults with congenital heart 24. Diller GP, Baumgartner H. Endocarditis in adults with con- disease. Heart 2017; 103: 1380-8. genital heart disease: new answers-new questions. Eur Heart J 45. Roca-Luque I, Rivas-Gándara N, Subirà LD, et al. Mecha- 2017; 38: 2057-9. nisms of intra-atrial re-entrant tachycardias in congenital 25. Kuijpers JM, Koolbergen DR, Groenink M, et al. Incidence, heart disease: types and predictors. Am J Cardiol 2018; 122: risk factors, and predictors of infective endocarditis in adult 672-82. congenital heart disease: focus on the use of prosthetic mate- 46. Mandalenakis Z, Rosengren A, Lappas G, et al. Atrial fibrilla- rial. Eur Heart J 2017; 38: 2048-56. tion burden in young patients with congenital heart disease. 26. Mylotte D, Rushani D, Therrien J, et al. Incidence, predictors, Circulation 2018; 137: 928-37. and mortality of infective endocarditis in adults with congeni- 47. Labombarda F, Hamilton R, Shohoudi A, et al. Increasing tal heart disease without prosthetic valves. Am J Cardiol prevalence of atrial fibrillation and permanent atrial arrhyth- 2017; 120: 2278-83. mias in congenital heart disease. J Am Coll Cardiol 2017; 70: 27. Moore B, Cao J, Kotchetkova I, Celermajer DS. Incidence, 857-65. predictors and outcomes of infective endocarditis in a con- 48. Yang H, Kuijpers JM, de Groot JR, et al. Impact of atrial ar- temporary adult congenital heart disease population. Int J rhythmias on outcome in adults with congenital heart disease. Cardiol 2017; 249: 161-5. Int J Cardiol 2017; 248: 152-4. 28. Østergaard L, Valeur N, Ihlemann N, et al. Incidence of infec- 49. Hernández-Madrid A, Paul T, Abrams D, et al. Arrhythmias in tive endocarditis among patients considered at high risk. Eur congenital heart disease: a position paper of the European Heart J 2018; 39: 623-9. Heart Rhythm Association (EHRA), Association for European 29. Tutarel O, Alonso-Gonzalez R, Montanaro C, et al. Infective Paediatric and Congenital Cardiology (AEPC), and the Euro- endocarditis in adults with congenital heart disease remains a pean Society of Cardiology (ESC) Working Group on lethal disease. Heart 2018; 104: 161-5. Grown-up Congenital Heart Disease, endorsed by HRS, 30. Kubota K, Soma K, Uehara M, et al. Combined surgical and PACES, APHRS, and SOLAECE. Europace 2018; 20: 1719- medical therapy for candida prosthetic endocarditis in a pa- 53. tient with repaired tetralogy of Fallot. Int Heart J 2018; 59: 50. Moore B, Yu C, Kotchetkova I, Cordina R, Celermajer DS. In- 877-80. cidence and clinical characteristics of sudden cardiac death in 31. Olsen M, Marino B, Kaltman J, et al. Myocardial infarction in adult congenital heart disease. Int J Cardiol 2018; 254: 101-6. adults with congenital heart disease. Am J Cardiol 2017; 120: 51. Lynge TH, Jeppesen AG, Winkel BG, et al. Nationwide study 2272-7. of sudden cardiac death in people with congenital heart de- 32. Bokma JP, Zegstroo I, Kuijpers JM, et al. Factors associated fects aged 0 to 35 years. Circ Arrhythm Electrophysiol 2018; with coronary artery disease and stroke in adults with con- 11: e005757. genital heart disease. Heart 2018; 104: 574-80. 52. Schuijt MTU, Blok IM, Zwinderman AH, et al. Mortality in 33. Broberg CS, Valente AM, Huang J, et al. Myocardial fibrosis pulmonary arterial hypertension due to congenital heart dis- and its relation to adverse outcome in transposition of the ease: serial changes improve prognostication. Int J Cardiol great arteries with a systemic right ventricle. Int J Cardiol 2017; 243: 449-53. 2018; 271: 60-5. 53. Probst J, Diller GP, Reinecke H, et al. Prevention of sudden 34. Ciep!ucha A, Trojnarska O, Kociemba A, et al. Clinical as- cardiac death in patients with tetralogy of Fallot: risk assess- pects of myocardial fibrosis in adults with Ebstein’s anomaly. ment and long term outcome. Int J Cardiol 2018; 269: 91-6. Heart Vessels 2018; 33: 1076-85. 54. Marelli AJ, Ionescu-Ittu R, Mackie AS, Guo L, Dendukuri N, 35. Lluri G, Renella P, Finn JP, Vorobiof G, Aboulhosn J, Deb A. Kaouache M. Lifetime prevalence of congenital heart disease Prognostic significance of left ventricular fibrosis in patients in the general population from 2000 to 2010. Circulation with congenital bicuspid aortic valve. Am J Cardiol 2017; 2014; 130: 749-56. 120: 1176-9. 55. Williams RG. Late causes of death after congenital heart de- 36. Miyazaki A, Sakaguchi H, Noritake K, et al. Interventricular fects: a population-based study from Finland. J Am Coll Car- dyssynchrony in a patient with a biventricular physiology and diol 2016; 68: 499-501. a systemic right ventricle. Heart Vessels 2017; 32: 234-9. 56. Spector LG, Menk JS, Knight JH, et al. Trends in long-term 37. Shiina Y, Inai K, Takahashi T, et al. Inter- and intra- mortality after congenital heart surgery. J Am Coll Cardiol ventricular dyssynchrony in the systemic right ventricle is a 2018; 71: 2434-46. surrogate marker of major cardiac events in mildly sympto- 57. Mandalenakis Z, Rosengren A, Skoglund K, Lappas G, matic patients. Heart Vessels 2018; 33: 1086-93. Eriksson P, Dellborg M. Survivorship in children and young 38. Hinton RB, Ware SM. Heart failure in pediatric patients with adults with congenital heart disease in Sweden. JAMA Intern congenital heart disease. Circ Res 2017; 120: 978-94. 2017; 177: 224-30. 39. Lal S, Kotchetkova I, Cao J, Jackson D, Cordina R, Celerma- 58. Bouma BJ, Mulder BJ. Changing landscape of congenital jer DS. Heart failure admissions and poor subsequent out- heart disease. Circ Res 2017; 120: 908-22. comes in adults with congenital heart disease. Eur J Heart 59. Fahed AC, Gelb BD, Seidman JG, Seidman CE. Genetics of Fail 2018; 20: 812-5. congenital heart disease: the glass half empty. Circ Res 2013; IntHeartJ Advance Publication ISL1 MUTATION IN DORV 9

112: 707-20. 2247-54. 60. Li YJ, Yang YQ. An update on the molecular diagnosis of 81. Ramond F, Duband S, Croisille P, et al. Expanding the car- congenital heart disease: focus on loss-of-function mutations. diac spectrum of Noonan syndrome with RIT1 variant: left Expert Rev Mol Diagn 2017; 17: 393-401. main coronary artery atresia causing sudden death. Eur J Med 61. Blue GM, Kirk EP, Giannoulatou E, et al. Advances in the Genet 2017; 60: 299-302. genetics of congenital heart disease: a clinician’s guide. J Am 82. Liu Y, Li B, Xu Y, Sun K. Mutation screening of GATA4 Coll Cardiol 2017; 69: 859-70. gene in CTD patients within Chinese Han population. Pediatr 62. Kodo K, Nishizawa T, Furutani M, et al. GATA6 mutations Cardiol 2017; 38: 506-12. cause human cardiac outflow tract defects by disrupting 83. Zhang M, Li FX, Liu XY, et al. MESP1 loss-of-function mu- semaphorin-plexin signaling. Proc Natl Acad Sci U S A tation contributes to double outlet right ventricle. Mol Med 2009; 106: 13933-8. Rep 2017; 16: 2747-54. 63. Schott JJ, Benson DW, Basson CT, et al. Congenital heart dis- 84. Liu S, Su Z, Tan S, et al. Functional analyses of a novel ease caused by mutations in the transcription factor NKX2-5. CITED2 nonsynonymous mutation in Chinese Tibetan pa- Science 1998; 281: 108-11. tients with congenital heart disease. Pediatr Cardiol 2017; 38: 64. Yagi H, Furutani Y, Hamada H, et al. Role of TBX1 in human 1226-31. del22q11.2 syndrome. Lancet 2003; 362: 1366-73. 85.GuoC,WangQ,WangY,et al. Exome sequencing reveals 65. Basson CT, Bachinsky DR, Lin RC, et al. Mutations in hu- novel IRXI mutation in congenital heart disease. Mol Med man TBX5 [corrected] cause limb and cardiac malformation Rep 2017; 15: 3193-7. in Holt-Oram syndrome. Nat Genet 1997; 15: 30-5. 86. Cristo F, Inácio JM, de Almeida S, et al. Functional study of 66. Kirk EP, Sunde M, Costa MW, et al. Mutations in cardiac T- DAND5 variant in patients with congenital heart disease and box factor gene TBX20 are associated with diverse cardiac laterality defects. BMC Med Genet 2017; 18: 77. pathologies, including defects of septation and valvulogenesis 87. Vaidya A, Flores SK, Cheng ZM, et al. EPAS1 mutations and and cardiomyopathy. Am J Hum Genet 2007; 81: 280-91. paragangliomas in cyanotic congenital heart disease. N Engl J 67. Jin SC, Homsy J, Zaidi S, et al. Contribution of rare inherited Med 2018; 378: 1259-61. and de novo variants in 2,871 congenital heart disease pro- 88. Cheng H, Dharmadhikari AV, Varland S, et al. Truncating bands. Nat Genet 2017; 49: 1593-601. variants in NAA15 are associated with variable levels of in- 68. Wang X, Charng WL, Chen CA, et al. Germline mutations in tellectual disability, autism spectrum disorder, and congenital ABL1 cause an autosomal dominant syndrome characterized anomalies. Am J Hum Genet 2018; 102: 985-94. by congenital heart defects and skeletal malformations. Nat 89. Bashamboo A, Eozenou C, Jørgensen A, et al. Loss of func- Genet 2017; 49: 613-7. tion of the nuclear receptor NR2F2, encoding COUP-TF2, 69. Liu X, Yagi H, Saeed S, et al. The complex genetics of hy- causes testis development and cardiac defects in 46,XX chil- poplastic left heart syndrome. Nat Genet 2017; 49: 1152-9. dren. Am J Hum Genet 2018; 102: 487-93. 70. Sanna-Cherchi S, Khan K, Westland R, et al. Exome-wide as- 90. Tokita MJ, Chen CA, Chitayat D, et al. De novo missense sociation study identifies GREB1L mutations in congenital variants in TRAF7 cause developmental delay, congenital kidney malformations. Am J Hum Genet 2017; 101: 789-802. anomalies, and dysmorphic features. Am J Hum Genet 2018; 71. Tan TY, Gonzaga-Jauregui C, Bhoj EJ, et al. Monoallelic 103: 154-62. BMP2 variants predicted to result in haploinsufficiency cause 91. Bjornsson T, Thorolfsdottir RB, Sveinbjornsson G, et al.A craniofacial, skeletal, and cardiac features overlapping those rare missense mutation in MYH6 associates with non- of 20p12 deletions. Am J Hum Genet 2017; 101: 985-94. syndromic coarctation of the aorta. Eur Heart J 2018; 39: 72. Huang RT, Wang J, Xue S, et al. TBX20 loss-of-function mu- 3243-9. tation responsible for familial tetralogy of Fallot or sporadic 92. Lüscher TF. Congenital heart and aortic disease: novel genetic persistent truncus arteriosus. Int J Med Sci 2017; 14: 323-32. causes, biomarkers, and effect of pregnancy. Eur Heart J 73. Li L, Wang J, Liu XY, et al. HAND1 loss-of-function muta- 2018; 39: 3149-51. tion contributes to congenital double outlet right ventricle. Int 93. Li RG, Xu YJ, Wang J, et al. GATA4 loss-of-function muta- J Mol Med 2017; 39: 711-8. tion and the congenitally bicuspid aortic valve. Am J Cardiol 74. Wang J, Hu XQ, Guo YH, et al. HAND1 loss-of-function mu- 2018; 121: 469-74. tation causes tetralogy of Fallot. Pediatr Cardiol 2017; 38: 94. Gharibeh L, Komati H, Bossé Y, et al. GATA6 regulates aortic 547-57. valve remodeling, and its haploinsufficiency leads to right-left 75. Hempel M, Casar Tena T, Diehl T, et al. Compound heterozy- type bicuspid aortic valve. Circulation 2018; 138: 1025-38. gous GATA5 mutations in a girl with hydrops fetalis, con- 95. Xu YJ, Di RM, Qiao Q, et al. GATA6 loss-of-function muta- genital heart defects and genital anomalies. Hum Genet 2017; tion contributes to congenital bicuspid aortic valve. Gene 136: 339-46. 2018; 663: 115-20. 76. Chen HX, Zhang X, Hou HT, et al. Identification of a novel 96. Qiao XH, Wang Q, Wang J, et al. A novel NR2F2 loss-of- and functional mutation in the TBX5 gene in a patient by function mutation predisposes to congenital heart defect. Eur screening from 354 patients with isolated ventricular septal J Med Genet 2018; 61: 197-203. defect. Eur J Med Genet 2017; 60: 385-90. 97. Vande Perre P, Zazo Seco C, Patat O, et al. 4q25 microdele- 77. Qiao XH, Wang F, Zhang XL, et al. MEF2C loss-of-function tion encompassing PITX2: a patient presenting with tetralogy mutation contributes to congenital heart defects. Int J Med of Fallot and dental anomalies without ocular features. Eur J Sci 2017; 14: 1143-53. Med Genet 2018; 61: 72-8. 78. Durbin MD, Cadar AG, Williams CH, et al. Hypoplastic left 98. Lu CX, Wang W, Wang Q, Liu XY, Yang YQ. A novel MEF2 heart syndrome sequencing reveals a novel NOTCH1 muta- C loss-of-function mutation associated with congenital double tion in a family with single ventricle defects. Pediatr Cardiol outlet right ventricle. Pediatr Cardiol 2018; 39: 794-804. 2017; 38: 1232-40. 99. Yokoyama R, Kinoshita K, Hata Y, et al. A mutant HCN4 79. Scott DA, Hernandez-Garcia A, Azamian MS, et al. Congeni- channel in a family with bradycardia, left bundle branch tal heart defects and left ventricular non-compaction in males block, and left ventricular noncompaction. Heart Vessels with loss-of-function variants in NONO. J Med Genet 2017; 2018; 33: 802-19. 54: 47-53. 100. Nijak A, Alaerts M, Kuiperi C, et al. Left ventricular non- 80. Xu YJ, Qiu XB, Yuan F, et al. Prevalence and spectrum of compaction with Ebstein anomaly attributed to a TPM1 muta- NKX2.5 mutations in patients with congenital atrial septal tion. Eur J Med Genet 2018; 61: 8-10. defect and atrioventricular block. Mol Med Rep 2017; 15: 101. Zhang E, Hong N, Chen S, et al. Targeted sequencing identi- IntHeartJ 10 WANG, ET AL Advance Publication

fies novel GATA6 variants in a large cohort of patients with 113. Bu L, Jiang X, Martin-Puig S, et al. Human ISL1 heart pro- conotruncal heart defects. Gene 2018; 641: 341-8. genitors generate diverse multipotent cardiovascular cell line- 102. Pulignani S, Vecoli C, Borghini A, Foffa I, Ait-Alì L, Andre- ages. Nature 2009; 460: 113-7. assi MG. Targeted next-generation sequencing in patients 114. Stevens KN, Hakonarson H, Kim CE, et al. Common vari- with non-syndromic congenital heart disease. Pediatr Cardiol ation in ISL1 confers genetic susceptibility for human con- 2018; 39: 682-9. genital heart disease. PLoS One 2010; 5: e10855. 103. Jaouadi A, Tabebi M, Abdelhedi F, et al. A novel TBX1 mis- 115. Luo ZL, Sun H, Yang ZQ, et al. Genetic variations of ISL1 sense mutation in patients with syndromic congenital heart associated with human congenital heart disease in Chinese defects. Biochem Biophys Res Commun 2018; 499: 563-9. Han people. Genet Mol Res 2014; 13: 1329-38. 104. Zhang M, Li FX, Liu XY, et al. TBX1 loss-of-function muta- 116. Ma L, Wang J, Li L, et al. ISL1 loss-of-function mutation tion contributes to congenital conotruncal defects. Exp Ther contributes to congenital heart defects. Heart Vessels 2019; Med 2018; 15: 447-53. 34: 658-68. 105. Shanshen E, Rosenberg J, Van Bergen AH. Identification of 117. Dodou E, Verzi MP, Anderson JP, Xu SM, Black BL. Mef2c novel congenital heart disease candidate genes using chromo- is a direct transcriptional target of ISL1 and GATA factors in some microarray. Pediatr Cardiol 2018; 39: 148-59. the anterior heart field during mouse embryonic development. 106. Li X, Shi L, Xu M, Zheng X, Yu Y, Jin J. RCAN1 mutation Development 2004; 131: 3931-42. and functional characterization in children with sporadic con- 118. Friedrich FW, Dilanian G, Khattar P, et al. A novel genetic genital heart disease. Pediatr Cardiol 2018; 39: 226-35. variant in the transcription factor Islet-1 exerts gain of func- 107. Pu T, Liu Y, Xu R, Li F, Chen S, Sun K. Identification of tion on myocyte enhancer factor 2C promoter activity. Eur J ZFPM2 mutations in sporadic conotruncal heart defect pa- Heart Fail 2013; 15: 267-76. tients. Mol Genet Genomics 2018; 293: 217-23. 119. Kodo K, Nishizawa T, Furutani M, et al. Genetic analysis of 108. Manheimer KB, Richter F, Edelmann LJ, et al. Robust identi- essential cardiac transcription factors in 256 patients with fication of mosaic variants in congenital heart disease. Hum non-syndromic congenital heart defects. Circ J 2012; 76: Genet 2018; 137: 183-93. 1703-11. 109. Gould RA, Aziz H, Woods CE, et al. ROBO4 variants predis- 120. Yuan F, Qiu ZH, Wang XH, et al. MEF2C loss-of-function pose individuals to bicuspid aortic valve and thoracic aortic mutation associated with familial dilated cardiomyopathy. aneurysm. Nat Genet 2019; 51: 42-50. Clin Chem Lab Med 2018; 56: 502-11. 110. Cai CL, Liang X, Shi Y, et al. Isl1 identifies a cardiac pro- 121. Moretti A, Lam J, Evans SM, Laugwitz KL. Biology of Isl1+ genitor population that proliferates prior to differentiation and cardiac progenitor cells in development and disease. Cell Mol contributes a majority of cells to the heart. Dev Cell 2003; 5: Life Sci 2007; 64: 674-82. 877-89. 122. Xu YJ, Wang ZS, Yang CX, et al. Identification and func- 111. Laugwitz KL, Moretti A, Lam J, et al. Postnatal isl1+ car- tional characterization of an ISL1 mutation predisposing to dioblasts enter fully differentiated cardiomyocyte lineages. dilated cardiomyopathy. J Cardiovasc Transl Res 2018. (doi: Nature 2005; 433: 647-53. 10.1007/s12265-018-9851-8). 112. Moretti A, Caron L, Nakano A, et al. Multipotent embryonic 123. Yan H, Yuan W, Velculescu VE, Vogelstein B, Kinzler KW. isl1+ progenitor cells lead to cardiac, smooth muscle, and en- Allelic variation in human gene expression. Science 2002; dothelial cell diversification. Cell 2006; 127: 1151-65. 297: 1143.