Genetic Mutation Analysis in Japanese Patients with Non-Syndromic Congenital Heart Disease

ORIGINAL ARTICLE

Genetic mutation analysis in Japanese patients with non-syndromic congenital heart disease

Akiko Yoshida1,2, Hiroko Morisaki1,2,3, Mai Nakaji1,3, Masataka Kitano4, Ki-sung Kim5, Koichi Sagawa6, Shiro Ishikawa6, Ichiro Satokata7, Yoshihide Mitani8, Hitoshi Kato5, Kenji Hamaoka9, Shigeyuki Echigo4, Isao Shiraishi4 and Takayuki Morisaki1,2,3

Congenital heart disease (CHD) is the most common birth defect occurring in humans and some transcriptional factors have been identified as causative. However, additional mutation analysis of these genes is necessary to develop effective diagnostic and medical treatment methods. We conducted sequence analysis of the coding regions of NKX2.5, GATA4, TBX1, TBX5, TBX20, CFC1 and ZIC3 in 111 Japanese patients with non-syndromic CHD and 9 of their relatives. All patient samples were also analyzed by multiplex ligation-dependent probe amplification using probes involved in chromosome deletion related to CHD. Five novel variations of TBX5, GATA4 and TBX20 were detected in 6 of the patients, whereas none were found in 200 controls. The TBX5 variation p.Pro108Thr, located in the T-box domain, was identified in a patient with tricuspid atresia, an exon–intron boundary variation of GATA4 (IVS4+5G4A) was detected in a Tetralogy of Fallot patient and an 8p23 microdeletion was detected in one patient with atrioventricular septal defect and psychomotor delay. A total of seven non-synonymous polymorphisms were found in the patients and controls. Accumulation of novel variations of genes involving the cardiac development may be required for better understanding of CHD. Journal of Human Genetics (2016) 61, 157–162; doi:10.1038/jhg.2015.126; published online 22 October 2015

INTRODUCTION medical treatment. In the present study, we assessed the contributions Congenital heart disease (CHD) is the most common birth defect of those gene mutations in Japanese patients with non- occurring in humans and observed in 4–50 per 1000 live births.1 CHD syndromic CHD. may result in part from a birth malformation with a related syndrome, although most cases are identified as an isolated defect without MATERIALS AND METHODS syndromic features.2 Although the etiology of non-syndromic CHD Patients remains largely unknown, some transcriptional factors, including We recruited 111 patients diagnosed with non-syndromic CHDs who were NKX2.5,3 GATA4,4 TBX1,5 TBX5,6,7 TBX20,8 CFC19 and ZIC3,10 treated at six hospitals in Japan, including National Cerebral and Cardio- have been identified as major contributors to CHD in some cases.11 vascular Center, Kyoto Prefectural University of Medicine University Hospital, The TBX1 and TBX5 were also identified as responsible gene for National Center for Child Health and Development, Niigata University Medical ’ syndromic disorders, TBX1 mutations are detected with DiGeorge and Dental Hospital, Fukuoka Children s Hospital and Mie University Hospital, syndrome/velocardiofacial syndrome phenotype without 22q11.2 from May 2008 to September 2011. Among them, 14 patients were familial cases with CHD including 1 who had a twin, whereas the others were sporadic microdeletion5 and TBX5 mutations were detected as causative cases. Nine relatives of four CHD patients also participated in this study. gene of Holt–Oram syndrome (HOS).6,7 However, in a few cases, In addition, 200 healthy individuals were studied as controls in our search for TBX112 TBX513,14 and mutations are found in patient with mutations or variations found in CHD patients. isolated CHD. The diagnosis of CHD was confirmed by pediatric cardiologists at each According to previous reports, the prevalence of CHD caused by a hospital. The patient clinical characteristics are shown in Supplementary genetic mutation of such cardiac transcription factors is relatively rare. Table 1. The most frequent clinical feature was ventricular septal defect However, further accumulation of mutation analysis findings is (VSD), followed by pulmonary stenosis. We excluded patients with syndromic needed for developing more effective methods for diagnosis and features such as Down syndrome, Williams syndrome and DiGeorge syndrome,

1Department of Bioscience and Genetics, National Cerebral and Cardiovascular Center Research Institute, Suita, Japan; 2Department of Medical Genetics, National Cerebral and Cardiovascular Center, Suita, Japan; 3Department of Molecular Pathophysiology, Osaka University Graduate School of Pharmaceutical Sciences, Suita, Japan; 4Department of Pediatric Cardiology, National Cerebral and Cardiovascular Center, Suita, Japan; 5Division of Cardiology, National Center for Child Health and Development, Tokyo, Japan; 6Department of Pediatric Cardiology, Fukuoka Children’s Hospital Medical Center, Fukuoka, Japan; 7Niigata University Graduate School of Medical and Dental Sciences, Niigata, Japan; 8Department of Pediatrics, Mie University Graduate School of Medicine, Tsu, Japan and 9Department of Pediatric Cardiology and Nephrology, Kyoto Prefectural University of Medicine Graduate School of Medical Sciences, Kyoto, Japan Correspondence: Dr T Morisaki, Department of Bioscience and Genetics, National Cerebral and Cardiovascular Center Research Institute, 5-7-1 Fujishirodai, Suita 565-8565, Japan. E-mail:[email protected] Received 27 April 2015; revised 1 September 2015; accepted 23 September 2015; published online 22 October 2015 Genetic mutation analysis in patients with CHD A Yoshida et al 158

Table 1 Summary of variations detected in 111 patients and phenotype of cardiac defect

Nucleotide Amino-acid Familial or Congenital Normal Allele Gene Position change change Location sporadic heart defect Reference control rate frequencya Prediction Programb

TBX5 Exon 4 c.322C4A p.Pro108Thr T-BOX domain Sporadic TA Novel 0/200 — S: Affect protein function (P = 0.03) Po: Probably damaging Pm: Neutral Exon 8 c.791G4A p.Arg264Lys — Sporadic VSD Novel 0/200 0.75% S: Tolerated (P = 0.86) Po: Probably damaging Pm: Neutral Exon 8 c.791G4A p.Arg264Lys — Sporadic VSD and PA TBX20 Exon 7 c.991A4Gp.Thr331Ala— Sporadic VSD Novel 0/200 0.37% S: Tolerated (P = 1.00) Po: Benign Pm: Neutral GATA4 IVS4+5 G4A —— Exon–intron Sporadic TOF Novel 0/200 0.12% MaxEntScan: 1.77 boundary Exon 2 c.431C4T p.Ala144Val — Familial PA and ASD Novel 0/200 — S: Tolerated (P = 0.13) Po: Possibly damaging Pm: Pathological

Abbreviations: ASD, atrial septal defect; PA, pulmonary atresia; TA, tricuspid atresia; TOF, tetralogy of fallot; VSD, ventricular septal defect. aAllele frequency Human Genetic Variation Browser. bPrediction Program: S, SIFT program; Po, Polyphen-2; Pm, PMut.

as well as patients with a structural chromosomal abnormality. This study was chromosome regions reported as CHD-related regions, which were 4q34-qter, approved by the ethics committee of each institution, and written informed 9q34.3, 17p13.3, 10p15, 8p23, 22q11 and 22q13. Genes in these chromosomal consent was obtained from the subjects or their parents, as appropriate, regions are shown in Supplementary Table 9. Samples shown to have a 22q11 beforehand. deletion (DiGeorge syndrome) were excluded from analysis. MLPA was performed according to the manufacturer’s protocol using 50 ng of DNA. Sequence analysis Amplified fragments were separated with a 3130 DNA analyzer (Applied Genomic DNA was prepared from blood leukocytes or Epstein-Barr virus Biosystems) and results were quantitatively analyzed using Gene Mapper immortalized lymphocytes. We performed sequence analysis for the following software (Applied Biosystems). Data analysis was performed using Coffalyser seven genes related to CHD: NKX2.5 (NM_004387), GATA4 (NM_002052), MLPA software (MRC Holland). TBX1 (NM_080647), TBX5 (NM_000192), TBX20 (NM_001077653), CFC1 (NM_032545) and ZIC3 (NM_003413). First, we analyzed all obtained samples Allele frequency for NKX2.5, GATA4, TBX1, TBX5 and TBX20,thenCFC1 and ZIC3 were We investigated the allele frequency of genetic variations causing non- analyzed in 28 patients diagnosed with heterotaxic abnormalities, such as synonymous amino-acid change in Japanese individuals identified in this transposition of great arteries (TGA), double-outlet right ventricle (DORV), study using the Human Genetic Variation Browser (http://www.genome.med. single ventricle (SV) or asplenia. Primers were designed to contain all coding kyoto-u.ac.jp/SnpDB), accessed on 18 November 2014. exons of these genes using Primer express (Applied Biosystems, Foster City, CA, USA) or based on previous reports (Supplementary Tables 2–8). Mutation prediction PCR assays were performed using genomic DNA at 20 or 40 ng. Depending When we found new genetic variations causing a non-synonymous amino-acid on the PCR fragment length, the Taq polymerase was either AmpliTaq Gold change, we determined whether those variations could be a pathogenic (Applied Biosystems), Platinum Taq DNA Polymerase (Invitrogen, Carlsbad, mutation using the SIFT (http://sift.bii.a-star.edu.sg/index.html), PolyPhen-2 CA, USA) or LA Taq HS (TAKARA, Kusatsu, Japan). PCR was performed with (http://genetics.bwh.harvard.edu/pph2/index.shtml), PMut (http://mmb.pcb. a total volume of 20 μl containing standard PCR buffer mix, according to the ub.es/PMut/) and MaxEntScan (http://genes.mit.edu/burgelab/maxent/Xmax- protocol of the manufacturer, and the polymerase. When a GC-rich fragment entscan_scoreseq.html) programs, each accessed on 5 December 2014. was amplified with AmpliTaq Gold or Platinum Taq DNA polymerase, dimethyl sulfoxide (Sigma-Aldrich, St Louis, MO, USA) or an enhancer Statistical analysis (Invitrogen) was added. PCR assays were performed using an initial denatura- Statistical analysis of the frequency of polymorphisms in patients and controls tion step at 95 °C for 1–9 min, then 38 cycles of denaturation at 95–98 °C for was performed using Stat View 5 (SAS Institute, Cary, NC, USA). 10–30 s, annealing at 55–62 °C for 60–90 s and extension at 68–72 °C for 60–90 s. Each individual PCR condition was dependent on the combination of RESULTS polymerase and primers. Thermal cycling was performed using a Gene Amp In the present study, we found five novel variations in TBX5, GATA4 PCR System (Applied Biosystems) or T GRADIENT Thermoblok device and TBX20 in the patient samples, but not in the control samples (Biometra, Göttingen, Germany). The PCR products were confirmed by (Table 1). electrophoresis on 1% agarose gels. The products were purified using Exo-Sap IT (United States Biochemical, Cleveland, OH, USA), a standard There were two variations, p.Pro108Thr and p.Arg264Lys, found in TBX5 sequencing protocol was used, and analysis was conducted using PRISM 3730 . The p.Pro108Thr variant, located in the T-box domain, changes and 3130 DNA analyzers (Applied Biosystems). amino-acid polar characteristics from hydrophobic to hydrophilic. Its location is near the DNA-binding site and we speculated that it affects Multiplex ligation-dependent probe amplification (MLPA) protein function, as the SIFT and Polyphen-2 programs indicated that All patient samples were further analyzed by MLPA with SALSA P250 DiGeorge it would ‘affect protein function’ or is ‘probably damaging’. However, probe mix (MHC Holland, Amsterdam, Netherlands). Probes covered the PMut program indicated it would be ‘neutral’.Thisvariantwas

Journal of Human Genetics Genetic mutation analysis in patients with CHD A Yoshida et al 159 not found in the Japanese genetic variation database. The p.Arg264Lys of the N-terminal region of GATA4. The patient with this variation variant was found in two unrelated patients with VSD as the common was the only case of familial CHD. In addition, an 8p23 microdeletion clinical feature, although its functional effect was not clear, as the was detected in 1 patient with atrioventricular septal defect (AVSD) change is not located in the T-box domain and was found in the and psychomotor delay (Table 2). There were no novel variations Japanese genetic variation database (allele frequency 0.75%). found in NKX2.5, CFC1 and ZIC3 among 111 patients with CHD and The TBX20 p.Thr331Ala variation was found in one patient with 28 patients with heterotaxic abnormalities. VSD and not detected in any of the controls, and also noted in the In addition to five non-synonymous variants identified only in the Japanese genetic variation database (allele frequency 0.37%). This patients shown above, seven non-synonymous polymorphisms were variation is not located in the T-box domain and three prediction found in both the CHD patient and control groups (TBX1: programs suggested that it could be benign. p.Gly310Ser; GATA4: p.Val380Met, p.Pro407Gln, and p.Val431Gly; There were two variations, IVS4+5G4A and p.Ala144Val, found in CFC1: p.Ala145Thr, p.Pro154Leu, and c.563-574del; Table 3). There GATA4 4 – .IVS4+5G A, an exon intron boundary variation, was was no significant difference between patients and controls for the fi identi ed in one patient with tetralogy of fallot (TOF). Although it allele frequencies of those polymorphisms. Among these variations, was noted in the Japanese genetic variation database (0.12%), it is p.Gly310Ser13 in TBX1, as well as p.Val380Met15, p.Pro407Gln15 and ’ located in the conserved 5 splice site and the prediction program p.Val431Gly (rs114944893) in GATA4, and p.Ala145Thr16 in CFC1 MaxEntScan suggested that it caused aberrant splicing. The have been reported, whereas p.Pro154Leu and c.563-574del in CFC1 p.Ala144Val variation is located in the transcription activator domain are not reported although were found in both patients and controls.

Table 2 Chromosome microdeletion of non-syndromic CHD DISCUSSION ﬁ TBX5 TBX20 GATA4 Deletion Included genes Congenital heart defect Reference In this study, we found ve variants in , and ,and an 8p23 microdeletion in the examined patients, but not in the control 8p23 GATA4 AVSD/psychomotor delay Wat et al.,35 subjects, whereas seven non-synonymous polymorphisms were found Devriendt et al.,34 in both patients and controls. These variations were located in the Abbreviations: AVSD, atrioventricular septal defect; CHD, congenital heart disease. genes as shown in Figure 1. We also identiﬁed two variations of TBX5

Table 3 Polymorphisms found both in patients and controls

Amino-acid dbSNP ID and Patient’s congenital Normal control Gene Nucleotide change change reference Patient rate heart defect rate Prediction programa P-valueb

TBX1 c.928G4A p.Gly310Ser Xu et al.12 7/111 SV and PS 13/200 S: Tolerated(P = 0.58) 40.9999 (6.3%) / 8q23 deletion:AVSD (6.5%) Po: Possibly damaging / TBX5 Pro108Thr Pm: Neutral (322C4A):TA /AS/TOF/TGA/SVAS and PS

GATA4 c.1138G4A p.Val380Met rs114868912 2/111 DORV and Hypoplastic 3/200 S: Tolerated(P = 0.30) 40.9999 Kodo et al.15 (1.8%) LV (1.5%) Po: Benign /CoA and VSD Pm: Neutral c.1220C4A p.Pro407Gln rs115099192 4/111 DORV and hypoplastic 2/200 S: Tolerated(P = 0.11) 0.1917 Kodo et al.15 (3.6%) LV (1.0%) Po: Possibly damaging /CoA and VSD Pm: Pathological /VSD/SV and PS c.1292T4G p.Val431Gly rs114944893 2/111 SV and PS/ASD 1/200 S: Affect protein function 0.2906 (1.8%) (0.5%) (P = 0.03) Po: Benign Pm: Pathological

CFC1 c.433G4A p.Ala145Thr rs62164861 1/28 TGA 2/200 S: Tolerated(P = 0.66) 0.3263 Ozcelik et al.16 (3.6%) (1.0%) Po: Benign Pm: Neutral c.461C4T p.Pro154Leu Novel 1/28 TGA 3/200 S: Tolerated(P = 1.00) 0.4101 (3.6%) (1.5%) Po: Benign Pm: Pathological c.563-574del — Novel 1/28 DORV 2/200 — 0.3263 ACCGCC (3.6%) (1.0%) TCCTGC

Abbreviations: AS, aortic stenosis; ASD, atrial septal defect; AVSD, atrioventricular septal defect; CoA, coarctation of the aorta; DORV, double-outlet right ventricle; LV, left ventricle; PS, pulmonary stenosis; SV, single ventricle; SVAS, supuravalvular aortic stenosis; TA, tricuspid atresia; TGA, transposition of great arteries; TOF, tetralogy of fallot; VSD, ventricular septal defect. aPrediction Program: S, SIFT program; Po, Polyphen-2; Pm, PMut. bFisher’s exact test.

Journal of Human Genetics Genetic mutation analysis in patients with CHD A Yoshida et al 160

in this study (p.Pro108Thr and p.Arg264Lys) only in the patient TBX20 mutations have been reported in patients with non- group. The p.Pro108Thr variant may have a pathogenic role in CHD, syndromic CHD such as ASD, and heart valve or cardiomyopathy as this variant is located in the T-box domain, and was not identified abnormalities, with most of pathogenic mutations located in the in the present controls or found in a Japanese genetic variation T-box domain.23,24 Previously, several studies tried to identify TBX20 database, whereas prediction programs did not suggest a consistent mutation in various CHD phenotype patients, and the mutation – pathogenicity. On the other hand, p.Arg264Lys is suggested to have no detection rate in these was 0.6–5.3%.8,23,25 28 We detected significant effect in individuals with CHD, as this change was not p.Thr331Ala, a TBX20 variation, in a patient with VSD, although it located in the T-box domain and found in the Japanese genetic was not located in the T-box domain and VSD is not a common TBX20 variation database with a certain allele frequency. clinical feature associated with mutations. In the previous TBX20 fi The TBX5 gene is responsible for HOS, which is characterized by study, missense mutation was identi ed in a patient with small 8 upper limb deformity and CHD (OMIM142900).6,7 The more VSD. Furthermore, this variant was assessed to be a non-pathogenic common cardiac defects were ASD, VSD and cardiac conduction variation by all of the prediction programs and detected in a Japanese TBX20 diseases.17 In a previous study, TBX5 mutations were detected in more genetic variation database. Therefore, the variation found in fi than 70% of examined patients17 when they met the criteria of HOS our study may not have a signi cant impact on VSD phenotype. including upper limb malformation and 70% of those mutations were GATA4 is thought to be an essential transcription factor in the developing heart. Until now, ~ 110 GATA4 mutations have been reported to cause haploinsufficiency.18 On the other hand, missense reported in CHD patients, particularly in association with a septal mutations have been found in patients with various phenotypes of defect such as atrial septal defect (ASD), VSD or AVSD.29 Mutation CHD and/or limb deformities, with symptoms ranging from mild to detection rate (0–12.5%) of GATA4 varied depending on each severe.17–20 Furthermore, patients with family members with the same study,29,30 and our detection rate 0.9% (1/111) was within the range mutation have been reported to show various phenotypes, such as of those results. We identified a novel GATA4 variation at the having only CHD, CHD with limb deformity or even no heart defect – 13 exon intron boundary of exon 4 (IVS4+5) in a TOF patient, which or limb deformity. Also, it was previously speculated that the gain of was not found in the 200 controls, although it was noted in a Japanese TBX5 function mutation in may result in a heart defect but not limb genetic variation database despite having a low allele frequency. An 14 deformity. Mutations detected in patients having only CHD in alteration of this position is thought to cause a splicing defect previous reports were located in both inside and outside of T-box due to impaired interaction of pre-mRNA with U1 and U6 13,14,21,22 domain. small nuclear RNAs,31 resulting in exon skipping or aberrant splicing In the present study, patients with TBX5 p.Pro108Thr or for exon 4. As exon 4 of GATA4 contains important zinc finger p.Arg264Lys showed no limb deformities and were diagnosed as domains, that would result in impairment of GATA4 as a transcription non-syndromic CHD. In addition, their cardiac defects (tricuspid factor. However, further studies are needed to confirm this, as we atresia; TA/VSD/pulmonary atresia; PA) are not considered to be could not confirm a functional defect of this variant. Another variant common in HOS. In the previous studies of TBX5 mutation analysis of GATA4 identified in our study, p.Ala144Val, found in a patient with in CHD patients, the TBX5 mutation was detected in 1–2% of the PA and ASD, was located in the transcriptional activation domain 2 patients; two mutations were detected in 94 patients with TOF14 (one (TAD2) of the GATA4 N-terminal region. Previous study reported patient with TOF showed also limb deformity including bilateral that variations located in TAD2 demonstrated no transcriptional triphalangeal thumbs and fifth finger clinodactyly) and two mutations activity change,32 whereas the other showed that transcriptional were identified in 78 patients with bicuspid valve.21 Therefore, the activity was not significantly altered but the DNA-binding affinity were variation detection rate (2.7%, 3/111) was similar to that in previous changed.29 In the present case, a study of the parents could not be reports. However, we could not simply compare these figures as CHD performed and the position of 144 alanine is not evolutionally phenotypes of each study were not the same of ours. conserved. Therefore, the functional relevance of the p.Ala144Val Although our patients with TBX5 variations may be atypical HOS, variant is not clear at this time. variations in TBX5 are considered to contribute to non-syndromic In our MLPA analysis, an 8p23 microdeletion was identified in a congenital heart defects. patient with AVSD and psychomotor delay. Large cohort study analyzing gene copy number spectra in 945 CHD patients showed 8p23 deletion including GATA4 in 3 patients.33 In other previous TBX5 p.Pro108Thr p.Arg264Lys reports,34,35 an 8p23 deletion was indicated to cause CHD (including

T-box AVSD), mental retardation, microcephaly, congenital diaphragmatic hernia and behavioral problems. Therefore, we think that CHD and other phenotypes in our patient might be due to that deletion. TBX20 p.Thr331Ala We also found several polymorphisms in candidate genes. The T-box TBX1 p.Gly310Ser variant was frequently found in both patients and controls, as previously noted.12 This variant was first reported as a GATA4 p.Ala144Val de novo ex4 IVS4+5G>A pathogenic mutation in a patient diagnosed with 22q11.2 5 TAD1 TAD2 ZF4 ZF NLS syndrome, although in whom a 22q11.2 deletion was not detected. However, the transcriptional activity of mutant TBX1 with Figure 1 Variants of TBX5, TBX20 and GATA4 are detected in this study. p.Gly310Ser was shown to have no significant change12,36 or only Schematic representation of variants of TBX5, TBX20 and GATA4 detected slightly increased.37 Another study indicated that Glycine310 is in this study is shown. Protein structure of TBX5 (518 amino acids), TBX20 38 (447 amino acids) and GATA4 (442 amino acids) was shown. ex4: exon 4; important for interaction with SMAD1, whereas a recent report NLS, nuclear localization signal; TAD1, transcriptional activation domain 1; stated that homozygous Ser310 was detected in a patient with severe 12 TAD2, transcriptional activation domain 2; ZF, Zinc finger DNA-binding CHD whose parents were heterozygous for Gly310Ser. Although it is domain. possible for Ser310 to be a pathogenic factor for CHD, patients who

Journal of Human Genetics Genetic mutation analysis in patients with CHD A Yoshida et al 161 are homozygous for p.Gly310Ser variant were not identified in 7 Li, Q. Y., Newbury-Ecob, R. A., Terrett, J. A., Wilson, D. I., Curtis, A. R., Yi, C. H. et al. our study. Holt-Oram syndrome is caused by mutations in TBX5, a member of the Brachyury (T) gene family. Nat. Genet. 15,21–29 (1997). GATA4 variants were found in both patients and controls, of which 8 Kirk, E. P., Sunde, M., Costa, M. W., Rankin, S. A., Wolstein, O., Castro, M. L. et al. two GATA4 variants, p.Val380Met and p.Pro407Gln, were previously Mutations in cardiac T-box factor gene TBX20 are associated with diverse cardiac 15 GATA4 pathologies, including defects of septation and valvulogenesis and cardiomyopathy. Am. reported as SNPs in Japanese. p.Val431Gly was found in the J. Hum. Genet. 81,280–291 (2007). database (rs114944893) and this position is not conserved with that in 9 Bamford, R. N., Roessler, E., Burdine, R. D., Saplakoğlu, U., dela Cruz, J., Splitt, M. other mammals. et al. Loss-of-function mutations in the EGF-CFC gene CFC1 are associated with human left-right laterality defects. Nat. Genet. 26,365–369 (2000). Our study also detected three polymorphisms in CFC1 10 Gebbia, M., Ferrero, G. B., Pilia, G., Bassi, M. T., Aylsworth, A., Penman-Splitt, M. et al. (p.Ala145Thr, p.Pro154Leu and deletion of c.563_574), which is a X-linked situs abnormalities result from mutations in ZIC3. Nat. Genet. 17, 305–308 (1997). member of the epidermal growth factor-Cripto/FRL-1/Cryptic 11 McCulley, D. J. & Black, B. L. Transcription factor pathways and congenital heart (EGF-CFC) gene family. The Cfc1 homozygous mutation mice show disease. Curr. Top. Dev. Biol. 100,253–277 (2012). 12 Xu, Y. J., Chen, S., Zhang, J., Fang, S. H., Guo, Q. Q., Wang, J. et al. Novel TBX1 visceral laterality and complicated heart malformations, such as TGA loss-of-function mutation causes isolated conotruncal heart defects in Chinese patients 39,40 and DORV. In other reports, p.Ala145Thr was evaluated as a without 22q11.2 deletion. BMC Med. Genet. 15, 78 (2014). variant of undetermined significance41 or a polymorphism detected in 13 Brassington, A. M., Sung, S. S., Toydemir, R. M., Le, T., Roeder, A. D., Rutherford, A. E. et al. Expressivity of Holt-Oram syndrome is not predicted by TBX5 16 controls. In this regard, our results suggest that p.Ala145Thr is a genotype. Am. J. Hum. Genet. 73,74–85 (2003). common polymorphism. In addition, we detected p.Pro154Leu as a 14 Baban, A., Postma, A. V., Marini, M., Trocchio, G., Santilli, A., Pelegrini, M. et al. Identification of TBX5 mutations in a series of 94 patients with Tetralogy of Fallot. novel polymorphism and consider that this variant has scant effect on Am. J. Med. Genet. A 164, 3100–3107 (2014). the function of the gene, as 154 proline is not conserved in other 15 Kodo, K., Nishizawa, T., Furutani, M., Arai, S., Ishihara, K., Oda, M. et al. Genetic species. Furthermore, deletion of the carboxy side (c.563-574) of CFC1 analysis of essential cardiac transcription factors in 256 patients with non-syndromic congenital heart defects. Circ. J. 76,1703–1711 (2012). was detected in one patient and two controls in this study. A previous 16 Ozcelik, C., Bit-Avragim, N., Panek, A., Gaio, U., Geier, C., Lange, P. E. et al. Mutations report showed that the carboxy side of the membrane associating in the EGF-CFC gene cryptic are an infrequent cause of congenital heart disease. Pediatr. Cardiol. 27,695–698 (2006). domain was not conserved in the EGF-CFC family, whereas 17 McDermott, D. A., Bressan, M. C., He, J., Lee, J. S., Aftimos, S., Brueckner, M. et al. p.Arg189Cys, included in the c.563-574deletion region, showed TBX5 genetic testing validates strict clinical criteria for Holt-Oram syndrome. Pediatr. 9 Res. 58,981–986 (2005). normal function in several assays. Our results also suggest that 18 Boogerd, C. J., Dooijes, D., Ilgun, A., Mathijssen, I. B., Hordijk, R., van de Laar, I. M. heterozygous deletion in the c.563_574 region has no appreciable et al. Functional analysis of novel TBX5 T-box mutations associated with Holt-Oram effect on susceptibility for CHD. syndrome. Cardiovasc. Res. 88,130–139 (2010). 19 Cross, S. J., Ching, Y. H., Li, Q. Y., Armstrong-Buisseret, L., Spranger, S., Lyonnet, S. Regarding the significance of the variants found in this study, there et al. The mutation spectrum in Holt-Oram syndrome. J. Med. Genet. 37, are some limitations for a full evaluation, as we could not obtain 785–787 (2000). 20 Basson, C. T., Huang, T., Lin, R. C., Bachinsky, D. R., Weremowicz, S., Vaglio, A. et al. samples from family members of patients with a detected variant. Different TBX5 interactions in heart and limb defined by Holt-Oram syndrome Additional functional analysis is needed to confirm the pathological mutations. Proc. Natl Acad. Sci. USA 96,2919–2924 (1999). significance of variants noted here. 21 Bonachea, E. M., Zender, G., White, P., Corsmeier, D., Newsom, D., Fitzgerald-Butt, S. et al. Use of a targeted, combinatorial next-generation sequencing approach for the study of bicuspid aortic valve. BMC Med. Genomics 7, 56 (2014). CONFLICT OF INTEREST 22 Fan, C., Duhagon, M. A., Oberti, C., Chen, S., Hiroi, Y., Komuro, I. et al. Novel TBX5 fl mutations and molecular mechanism for Holt-Oram syndrome. J. Med. Genet. 40, The authors declare no con ict of interest. e29 (2003). 23 Posch, M. G., Gramlich, M., Sunde, M., Schmitt, K. R., Lee, S. H., Richter, S. et al. A ACKNOWLEDGEMENTS gain-of-function TBX20 mutation causes congenital atrial septal defects, patent – fi foramen ovale and cardiac valve defects. J. Med. Genet. 47,230 235 (2010). This work was supported by a Grant-in-Aid for Scienti c Research from the 24 Posch, M. G., Perrot, A., Berger, F. & Ozcelik, C. Molecular genetics of congenital atrial Ministry of Education, Culture, Sports, Science and Technology of Japan, and septal defects. Clin. Res. Cardiol. 99,137–147 (2010). grants from the Japan Science and Technology Corporation, Ministry of Health, 25 Qian, L., Mohapatra, B., Akasaka, T., Liu, J., Ocorr, K., Towbin, J. A. et al. Transcription Labour and Welfare of Japan and the Program for Promotion of Fundamental factor neuromancer/TBX20 is required for cardiac function in Drosophila with implications for human heart disease. Proc. Natl Acad. Sci. USA 105, Studies in Health Sciences of the National Institute of Biomedical Innovation 19833–19838 (2008). (NIBIO). 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