The T-box family transcription factor gene TBX20 acts in a conserved regulatory network, guiding heart formation and patterning in diverse species. Mouse Tbx20 is expressed in cardiac progenitor cells, differentiating cardiomyocytes, and developing valvular tissue, and its deletion or RNA interference–mediated knockdown is catastrophic for heart development. TBX20 interacts physically, functionally, and genetically with other cardiac transcription factors, including NKX2-5, GATA4, and TBX5, mutations of which cause congenital heart disease (CHD). Here, we report nonsense (Q195X) and missense (I152M) germline mutations within the T-box DNA-binding domain of human TBX20 that were associated with a family history of CHD and a complex spectrum of developmental anomalies, including defects in septation, chamber growth, and valvulogenesis. Biophysical characterization of wild-type and mutant proteins indicated how the missense mutation disrupts the structure and function of the TBX20 T-box. Dilated cardiomyopathy was a feature of the TBX20 mutant phenotype in humans and mice, suggesting that mutations in developmental transcription factors can provide a sensitized template for adult-onset heart disease. Our findings are the first to link TBX20 mutations to human pathology. They provide insights into how mutation of different genes in an interactive regulatory circuit lead to diverse clinical phenotypes, with implications for diagnosis, genetic screening, and patient follow-up. The T-box family transcription factor gene TBX20 acts in a conserved regulatory network, guiding heart formation and patterning in diverse species. Mouse Tbx20 is expressed in cardiac progenitor cells, differentiating cardiomyocytes, and developing valvular tissue, and its deletion or RNA interference–mediated knockdown is catastrophic for heart development. TBX20 interacts physically, functionally, and genetically with other cardiac transcription factors, including NKX2-5, GATA4, and TBX5, mutations of which cause congenital heart disease (CHD). Here, we report nonsense (Q195X) and missense (I152M) germline mutations within the T-box DNA-binding domain of human TBX20 that were associated with a family history of CHD and a complex spectrum of developmental anomalies, including defects in septation, chamber growth, and valvulogenesis. Biophysical characterization of wild-type and mutant proteins indicated how the missense mutation disrupts the structure and function of the TBX20 T-box. Dilated cardiomyopathy was a feature of the TBX20 mutant phenotype in humans and mice, suggesting that mutations in developmental transcription factors can provide a sensitized template for adult-onset heart disease. Our findings are the first to link TBX20 mutations to human pathology. They provide insights into how mutation of different genes in an interactive regulatory circuit lead to diverse clinical phenotypes, with implications for diagnosis, genetic screening, and patient follow-up. Structural malformations of the heart (congenital heart disease [CHD]) are extremely common, present in nearly 1 in 100 live births and 1 in 10 stillborns. Treatment of CHD often involves highly invasive surgery in childhood, conferring a major economic burden on health resources and a life-long emotional burden for affected individuals and families. A subset of CHD is familial, and, in some cases, causative genes have been identified, most encoding cardiac transcription factors including NKX2-5 (MIM 600584), GATA4 (MIM 600576), and TBX5 (MIM 601620).1Gruber PJ Epstein JA Development gone awry: congenital heart disease.Circ Res. 2004; 94: 273-283Crossref PubMed Scopus (105) Google Scholar These factors are part of a conserved regulatory network that controls cardiogenesis in species as diverse as man and insects.2Harvey RP NK-2 homeobox genes and heart development.Dev Biol. 1996; 178: 203-216Crossref PubMed Scopus (477) Google Scholar, 3Cripps RM Olson E Control of cardiac development by an evolutionarily conserved transcriptional network.Dev Biol. 2002; 246: 14-28Crossref PubMed Scopus (256) Google Scholar However, dominant mutations in cardiac developmental transcription factors account thus far for only a minority of familial cases and for few isolated cases of CHD.4Elliott DA Kirk E Yeoh T Chander S McKenzie F Taylor P Grossfeld P Fatkin D Jones O Hayes P et al.Cardiac homeobox gene NKX2-5 mutations and congenital heart disease: associations with atrial septal defect and hyperplastic left heart syndrome.J Am Coll Cardiol. 2003; 41: 2072-2076Abstract Full Text Full Text PDF PubMed Scopus (194) Google Scholar, 5McElhinney DB Geiger E Blinder J Benson W Goldmuntz E NKX2.5 mutations in patients with congenital heart disease.J Am Coll Cardiol. 2003; 42: 1650-1655Abstract Full Text Full Text PDF PubMed Scopus (291) Google Scholar Therefore, a major imperative in this field remains to dissect cardiac developmental pathways in detail and to understand how mutations in genes encoding the various components of these pathways cause CHD at the genetic and mechanistic levels. T-box transcription factors are characterized by the presence of a highly conserved, 180-aa, sequence-specific DNA-binding domain termed the “T-box.” These factors act as transcriptional activators and repressors and are known to function in a combinatorial and hierarchical fashion in many developmental processes.6Stennard FA Harvey RP T-box transcription factors and their roles in regulatory hierarchies in the developing heart.Development. 2005; 132: 4897-4910Crossref PubMed Scopus (125) Google Scholar At least seven members of the T-box gene family are expressed in the developing heart in humans and vertebrate models.6Stennard FA Harvey RP T-box transcription factors and their roles in regulatory hierarchies in the developing heart.Development. 2005; 132: 4897-4910Crossref PubMed Scopus (125) Google ScholarTBX1 (MIM 602054) is deleted in 22q11 deletion syndrome (MIM 188400 and 192430), the most common genetic deletion syndrome in humans, and has emerged as the leading candidate for causation of the complex cardiac and pharyngeal malformations that constitute the syndrome.7Yamagishi H Srivastava D Unravelling the genetic and developmental mysteries of 22q11 deletion syndrome.Trends Mol Med. 2003; 9: 383-389Abstract Full Text Full Text PDF PubMed Scopus (100) Google Scholar TBX1 has multiple roles in pharyngeal development, including, in mouse, regulation of the Fgf8 gene (GenBank accession number NM_010205), involved in maintenance and growth of neural crest cells and an anterior heart progenitor population (the anterior second heart field) that contributes cardiomyocytes, smooth muscle, and endothelial cells to the outflow tract.6Stennard FA Harvey RP T-box transcription factors and their roles in regulatory hierarchies in the developing heart.Development. 2005; 132: 4897-4910Crossref PubMed Scopus (125) Google Scholar, 8Buckingham ME Meilhac S Zaffran S Building the mammalian heart from two sources of myocardial cells.Nat Rev Genet. 2005; 6: 826-835Crossref PubMed Scopus (848) Google Scholar Mutations in TBX5 cause the rare autosomal dominant Holt-Oram syndrome (MIM 142900), characterized by congenital forelimb and cardiac malformations, the latter including atrial septal defect (ASD), ventricular septal defect (VSD), tetralogy of Fallot, hypoplastic left heart, and conduction abnormalities.9Basson CT Bachinsky DR Lin RC Levi T Elkins JA Soults J Grayzel D Kroumpouzou E Traill TA Leblanc-Straceski J et al.Mutations in human Tbx5 cause limb and cardiac malformation in Holt-Oram syndrome.Nat Genet. 1997; 15: 30-35Crossref PubMed Scopus (865) Google Scholar, 10Li QY Newbury-Ecob RA Terrett JA Wilson DI Curtis ARJ Yi CH Gebuhr T Bullen PJ Robson SC Strachan T et al.Holt-Oram syndrome is caused by mutations in TBX5, a member of the Brachyury (T) gene family.Nat Genet. 1997; 15: 21-29Crossref PubMed Scopus (716) Google ScholarTbx2 (GenBank accession number NM_009324), Tbx3 (GenBank accession numbers NM_198052 and NM_011535), and Tbx18 (GenBank accession number NM_023814) are involved in cardiac chamber and inflow-tract development, respectively, in mice,6Stennard FA Harvey RP T-box transcription factors and their roles in regulatory hierarchies in the developing heart.Development. 2005; 132: 4897-4910Crossref PubMed Scopus (125) Google Scholar, 11Christoffels VM Mommersteeg MT Trowe MO Prall OWJ de Gier-de Vries C Soufan AT Bussen M Schuster-Gossler K Harvey RP Moorman AF et al.Formation of the venous pole of the heart from an Nkx2-5-negative precursor population requires Tbx18.Circ Res. 2006; 98: 1555-1563Crossref PubMed Scopus (237) Google Scholar and, although TBX3 (MIM 601620) is mutated in ulnar-mammary syndrome,12Bamshad M Lin RC Law DJ Watkins WS Krakowiak PA Moore ME Franceschini P Lala R Holmes LB Gebuhr TC et al.Mutations in human TBX3 alter limb apocrine and genital development in ulnar-mammary syndrome.Nat Genet. 1997; 16: 311-315Crossref PubMed Scopus (430) Google Scholar these genes have not thus far been implicated in CHD in humans. TBX20 (MIM 606061) is an ancient member of the T-box superfamily related to TBX1, and the expression and function of the Tbx20 gene (GenBank accession number NM_020496) has recently been characterized in a number of models.6Stennard FA Harvey RP T-box transcription factors and their roles in regulatory hierarchies in the developing heart.Development. 2005; 132: 4897-4910Crossref PubMed Scopus (125) Google Scholar, 13Zaffran S Reim I Qian L Lo PC Bodmer R Frasch M Cardioblast-intrinsic Tinman activity controls proper diversification and differention of myocardial cells in Drosophila.Development. 2006; 133: 4073-4083Crossref PubMed Scopus (70) Google Scholar, 14Brown DD Martz SN Binder O Goetz SC Price BMJ Smith J Conlon FL Tbx5 and Tbx20 act synergistically to control vertebrate heart morphogenesis.Development. 2005; 132: 553-563Crossref PubMed Scopus (110) Google Scholar, 15Szeto DP Griffin KJ Kimmelman DK hrT is required for cardiovascular development in zebrafish.Development. 2002; 129: 5093-5101PubMed Google Scholar In mice, Tbx20 is expressed in cardiac progenitor cells, as well as in the developing myocardium and endothelial cells associated with endocardial cushions, the precursor structures for the cardiac valves and the atrioventricular septum.16Stennard FA Costa MW Elliott DA Rankin S Haast SJP Lai D McDonald LPA Niederreither K Dolle P Bruneau BG et al.Cardiac T-box factor Tbx20 directly interacts with Nkx2-5, GATA4 and GATA5 in regulation of gene expression in the developing heart.Dev Biol. 2003; 262: 206-224Crossref PubMed Scopus (223) Google Scholar Tbx20 carries strong transcriptional activation and repression domains, and it physically or genetically interacts with other cardiac developmental transcription factors, including Nkx2-5 (GenBank accession number NM_008700), Gata4 (GenBank accession number DQ436915), Gata5 (GenBank accession number NM_008093), and Tbx5 (GenBank accession number NM_011537).14Brown DD Martz SN Binder O Goetz SC Price BMJ Smith J Conlon FL Tbx5 and Tbx20 act synergistically to control vertebrate heart morphogenesis.Development. 2005; 132: 553-563Crossref PubMed Scopus (110) Google Scholar, 16Stennard FA Costa MW Elliott DA Rankin S Haast SJP Lai D McDonald LPA Niederreither K Dolle P Bruneau BG et al.Cardiac T-box factor Tbx20 directly interacts with Nkx2-5, GATA4 and GATA5 in regulation of gene expression in the developing heart.Dev Biol. 2003; 262: 206-224Crossref PubMed Scopus (223) Google Scholar Loss of Tbx20 in mice is catastrophic for heart development. Homozygous mutants show a rudimentary heart that is poorly proliferative and lacks chamber myocardium and in which expression of the early transcription factor network is compromised.17Stennard FA Costa MW Lai D Biben C Furtado M Solloway MJ McCulley DJ Leimena C Preis JI Dunwoodie SL et al.Murine T-box transcription factor Tbx20 acts as a repressor during heart development, and is essential for adult heart integrity, function and adaptation.Development. 2005; 132: 2451-2462Crossref PubMed Scopus (166) Google Scholar, 18Cai CL Zhou W Yang L Bu L Qyang Y Zhang X Li X Rosenfeld MG Chen J Evans S T-box genes coordinate regional rates of proliferation and regional specification during cardiogenesis.Development. 2005; 132: 2475-2487Crossref PubMed Scopus (174) Google Scholar, 19Singh MK Christoffels VM Dias JM Trowe MO Petry M Schuster-Gossler K Burger A Ericson J Kispert A Tbx20 is essential for cardiac chamber differentiation and repression of Tbx2.Development. 2005; 132: 2697-2707Crossref PubMed Scopus (158) Google Scholar Tbx20 appears to directly repress another T-box gene, Tbx2, which is itself a repressor involved in allocation of chamber and nonchamber myocardium in the early heart tube. A partial knockdown of Tbx20 expression with RNA interference (RNAi) technology20Takeuchi JJ Mileikovskaia M Koshiba-Takeuchi K Heidt AB Mori AD Arruda EP Gertsensein M Georges R Davidson L Mo R et al.Tbx20 dose-dependently regulates transcription factor networks required for mouse heart and motorneuron development.Development. 2005; 132: 2463-2474Crossref PubMed Scopus (177) Google Scholar and analysis of Tbx20 function with use of a chick atrioventricular canal explant system21Shelton EL Yutzey KE Tbx20 regulation of endocardial cushion proliferation and extracellular matrix gene expression.Dev Biol. 2007; 302: 376-388Crossref PubMed Scopus (85) Google Scholar have revealed later functions for Tbx20 in atrioventricular valve development. Adult heterozygous Tbx20-knockout mice show mild atrial septal abnormalities, including an increased prevalence of patent foramen ovale (PFO) and aneurysmal atrial septum primum, as well as mild dilated cardiomyopathy (DCM) and a genetic predisposition to frank ASD.17Stennard FA Costa MW Lai D Biben C Furtado M Solloway MJ McCulley DJ Leimena C Preis JI Dunwoodie SL et al.Murine T-box transcription factor Tbx20 acts as a repressor during heart development, and is essential for adult heart integrity, function and adaptation.Development. 2005; 132: 2451-2462Crossref PubMed Scopus (166) Google Scholar The essential roles of Tbx20 in heart development and adult heart function in mice raise the possibility that mutations in human TBX20 (Ensembl Genome Browser [chromosome 7p14.2] accession number ENSG00000164532) contribute to CHD. We therefore screened 352 CHD-affected probands for TBX20 mutations and found one missense and one nonsense mutation in probands with a family history of CHD. Mutations lay within exons encoding the T-box DNA-binding domain, and we provide structural, functional, and biophysical evidence of their deleterious action. TBX20 mutations were associated with a complex spectrum of developmental and functional abnormalities, including defects in septation, valvulogenesis, and chamber growth and cardiomyopathy. The discovery of CHD mutations in an additional gene functioning in the conserved cardiac regulatory network highlights the importance of this network in development and evolution and as a molecular target in cardiac pathology. Patients with CHD were unrelated individuals recruited without reference to family history during 2000–2006 from St. Vincent’s Hospital, Sydney Children’s Hospital, and The Children’s Hospital at Westmead, Sydney. Clinical evaluation by a cardiologist included medical history, 12-lead electrocardiography and transthoracic echocardiography, and/or transesophageal echocardiography (TEE). Diagnostic categorization of patients with CHD was made according to their most significant structural lesion. For example, a patient with an ASD and a left superior vena cava (SVC) would be categorized as having “ASD with other CHD” (see table 1). A patient with transposition of the great arteries and an ASD would be classified as having “other CHD,” since the transposition may represent more significant pathology. Ethnicity was determined by questionnaire. Informed written consent was obtained from all recruited patients. Study protocols were approved by the human research ethics committees of participating hospitals. The majority of white control individuals were unrelated anonymized individuals for whom atrial and ventricular septal status was undetermined. However, this group also included >100 “TEE controls,” who were unrelated individuals recruited from St. Vincent’s Hospital for whom TEE was performed for a number of indications and for whom ASD, VSD, and PFO were specifically excluded using intravenous saline contrast injection during the strain and release phases of the Valsalva maneuver. Mutation screening was also performed for a supplementary cohort of 90 probands with adult-onset familial DCM from St. Vincent’s Hospital or referred by collaborating physicians.Table 1Patient Cohort DetailsPhenotype(s)ASD OnlyaThree adults had mitral valve prolapse.ASD and Other CHDbIncluding sinus venosus ASD (n=13; all others are secundum ASD); partial anomalous pulmonary venous connection (n=6); left SVC (n=2); valvular lesions (n=5), including one example of supravalvar mitral ring; and coarctation of the aorta (n=1).VSD OnlyVSD and Other CHDcIncluding ASD (n=7), left SVC (n=5), aortic valve abnormalities (n=5), coarctation of the aorta (n=4), double-chambered right ventricle (n=2), pulmonary stenosis (n=1), patent ductus arteriosus (n=1), and partial anomalous venous connection (n=1). One subject had mitral valve prolapse, and one had supravalvar mitral ring.Other CHDdIncluding outflow tract lesions (n=75), atrioventricular septal defect and variants (n=18), functional single ventricle (n=17, including 2 with mitral valve atresia), heterotaxy (n=2), cor triatriatum (n=1), and Ebstein anomaly (n=1).No. of Subjects: Total151244122115 Male5316231070 With positive family historyePositive family history was defined as at least one first-degree relative affected with CHD. Thirty-seven subjects were found to have syndromes known to be associated with CHD, including trisomy 21 (n=20) and 22q microdeletions (n=12). However, only two subjects with a positive family history were from this group.205428 With AV conduction blockfFirst-degree or complete heart block. Complete and partial right bundle-branch block were not included in this group. Two subjects with ASD had left bundle-branch block.53010 With atrial fibrillation80000 With LV dysfunction5gAll subjects were aged >55 years. Subjects had normal LV size and contractility but impaired diastolic relaxation (n=2) or impaired systolic function with (n=2) or without (n=1) LV dilation.1hThis patient (family 2, individual III:4) was positive for TBX20 mutation Q195X.000Mean (range) age at enrollment, in years26 (0–79)12 (.2–62)6 (0–68)6 (0–59)4 (0–16)a Three adults had mitral valve prolapse.b Including sinus venosus ASD (n=13; all others are secundum ASD); partial anomalous pulmonary venous connection (n=6); left SVC (n=2); valvular lesions (n=5), including one example of supravalvar mitral ring; and coarctation of the aorta (n=1).c Including ASD (n=7), left SVC (n=5), aortic valve abnormalities (n=5), coarctation of the aorta (n=4), double-chambered right ventricle (n=2), pulmonary stenosis (n=1), patent ductus arteriosus (n=1), and partial anomalous venous connection (n=1). One subject had mitral valve prolapse, and one had supravalvar mitral ring.d Including outflow tract lesions (n=75), atrioventricular septal defect and variants (n=18), functional single ventricle (n=17, including 2 with mitral valve atresia), heterotaxy (n=2), cor triatriatum (n=1), and Ebstein anomaly (n=1).e Positive family history was defined as at least one first-degree relative affected with CHD. Thirty-seven subjects were found to have syndromes known to be associated with CHD, including trisomy 21 (n=20) and 22q microdeletions (n=12). However, only two subjects with a positive family history were from this group.f First-degree or complete heart block. Complete and partial right bundle-branch block were not included in this group. Two subjects with ASD had left bundle-branch block.g All subjects were aged >55 years. Subjects had normal LV size and contractility but impaired diastolic relaxation (n=2) or impaired systolic function with (n=2) or without (n=1) LV dilation.h This patient (family 2, individual III:4) was positive for TBX20 mutation Q195X. Open table in a new tab TBX20 coding exons were amplified by PCR from 100 ng of leukocyte DNA, were purified with PCR Cleanup Plates (Millipore), and were sequenced using Big Dye Terminator v3.1 kit (Applied Biosystems) and ABI PRISM 3700 DNA Analyzer. Transfection assays and frog-embryo mRNA microinjection assays were performed as described elsewhere,16Stennard FA Costa MW Elliott DA Rankin S Haast SJP Lai D McDonald LPA Niederreither K Dolle P Bruneau BG et al.Cardiac T-box factor Tbx20 directly interacts with Nkx2-5, GATA4 and GATA5 in regulation of gene expression in the developing heart.Dev Biol. 2003; 262: 206-224Crossref PubMed Scopus (223) Google Scholar except that, in the 293T-cell assay exploring Tbx20c function, Tbx20c plasmids were cotransfected with an expression plasmid encoding Sumo-1 (GenBank accession number NM_009460),22Kerscher O Felberbaum R Hochstasser M Modification of proteins by ubiquitin and ubiquitin-like proteins.Annu Rev Cell Dev Biol. 2006; 22: 159-180Crossref PubMed Scopus (1145) Google Scholar which stimulated activity, although it is not known whether Tbx20 itself is sumoylated. Transcription data presented represent experiments performed in triplicate. Statistical analysis was performed using Student’s two-tailed t-test. A homology model of the T-box from mouse Tbx20 bound to DNA was produced using the program SWISS-MODEL, with use of the crystal structure of the T-box from human TBX3 (Protein Data Bank ID 1h6f)23Coll M Seidman JG Muller CW Structure of the DNA-bound T-box domain of human TBX3, a transcription factor responsible for ulnar-mammary syndrome.Structure. 2002; 10: 343-356Abstract Full Text Full Text PDF PubMed Scopus (83) Google Scholar as template. The model structure was not subjected to energy minimization. Graphics were generated using PyMol and Adobe Illustrator CS2. Wild-type (WT) and I152M and Q195X Tbx20 proteins were prepared as glutathione S-transferase (GST) fusion proteins with use of a pGEX-4T-2 protein–expression vector in Escherichia coli BL21 (DE3) Rosetta cells (Novagen and Merck). Protein expression was induced by the addition of 0.4 mM isopropyl β-d-1-thiogalactopyranoside at OD600 of 0.6 and was continued at 22°C for 18 h. Cell pellets were resuspended in 20 mM 3-(N-Morpholino)propane sulphonic acid (MOPS) (pH 7.5), 150 mM NaCl, and 1 mM dithiothreitol (DTT) containing Complete Protease Inhibitors (Roche). Cells were lysed by sonication and then were treated with DNase I (Roche). Cell debris and inclusion bodies removed after centrifugation were solubilized in 8 M urea and were analyzed by SDS PAGE. DNA was precipitated from the lysate supernatant by addition of polyethyleneimine (0.1%), and fusion proteins were purified from the supernatant by affinity chromatography (Glutathione Sepharose 4B [Amersham Biosciences]) according to the manufacturer’s protocols. T-box domains were cleaved from GST on beads with thrombin and were further purified by cation-exchange chromatography on a UnoS.1 column (BioRad) running in 20 mM MOPS (pH 7.5), 1 mM DTT, and 10 μM ZnSO4, with a gradient of NaCl to remove any nucleic acids that remained associated with the protein throughout the affinity purification and cleavage steps. Q195X Tbx20 was unstable and formed inclusion bodies. Circular dichroism (CD) spectropolarimetry data were recorded on a Jasco J-720 spectropolarimeter equipped with a Neslab RTE-111 temperature controller. Far-UV CD spectra were collected at 20°C with a 1-mm cuvette, over the wavelength range 190–250 nm and with a speed of 20 nm/min, resolution of 0.5 nm, band width of 1 nm, and response time of 1 s. Final spectra were the average of three scans, corrected by subtracting a buffer-only spectrum. Protein concentration was estimated from A280, with use of a molar extinction coefficient of 21,430 M−1cm−1 (calculated from sequence data). Melting temperature (MT) was taken as the midpoint in the thermal denaturation curve, determined as the loss in secondary structure in the far-UV CD spectrum. Thermal data were collected at 215 nm, heating from 20°C to 80°C at 1°C per min, with a step size of 0.5°C, band width of 1 nm, and response time of 1 s. Protein concentration was 0.32 mg/ml for the far-UV spectra and 0.57 mg/ml for the thermal melt experiments, in 10 mM sodium phosphate and 150 mM NaF (pH 7.4). The MT was determined by fitting data to a sigmoidal function with use of the nonlinear least-squares fitter in MicroCal Origin. Surface plasmon-resonance analysis was performed on a Biacore 2000 SPR. A biotinylated double-stranded oligonucleotide corresponding to the T-half site16Stennard FA Costa MW Elliott DA Rankin S Haast SJP Lai D McDonald LPA Niederreither K Dolle P Bruneau BG et al.Cardiac T-box factor Tbx20 directly interacts with Nkx2-5, GATA4 and GATA5 in regulation of gene expression in the developing heart.Dev Biol. 2003; 262: 206-224Crossref PubMed Scopus (223) Google Scholar was immobilized on a streptavidin-coated SA sensor chip (Biacore). Single-stranded oligonucleotides (5′-biotin-ctcttataggtgtgaaaaccgtg-3′ and 5′-cacggttttcacacctata-3′) were annealed before binding. The buffer used for all experiments was 50 mM MOPS, 150 mM NaCl, 1 mM DTT, 0.005% P20 surfactant, and 10 μM ZnSO4. The chip was pretreated according to the manufacturer’s instructions, with conditioning solution (3×100 μl injections at 50 μl/min with 50 mM NaOH and 1 M NaCl). Each biotinylated double-stranded oligonucleotide was diluted to 2 nM in 50 mM MOPS (pH 7.4), 500 mM NaCl, 1 mM DTT, 0.005% (v/v) P20 surfactant, and 10 μM ZnSO4 and was injected into one of the sensor-chip channels at a flow rate of 10 μl/min for 10 min, resulting in an immobilization level of ∼600 response units (RUs). The sensor chip was then washed with 50 mM MOPS, 150 mM NaCl, 1 mM DTT, 0.005% P20 surfactant, and 10 μM ZnSO4. Upstream, unmodified channel surfaces were used for reference subtraction. Kinetic measurements were performed at 20°C with a KINJECT protocol and a flow rate of 30 μl/min in the same buffer, with increasing protein concentrations across the range 0.1–10 μM. Ninety microliters of each protein concentration was injected and, at the end of the association phase, was replaced with continuous buffer flow, to monitor dissociation kinetics. WT and mutant protein samples were sampled alternately, zero-concentration samples were included for double referencing, and three cycles were performed. Data analysis was performed with the BIA evaluation software. For one-dimensional 1H nuclear magnetic resonance (NMR) spectra of WT and mutant Tbx20 T-box domains, proteins were prepared at 9.8 mg/ml (WT) and 1.3 mg/ml (I152M) in 50 mM MOPS (pH 7.5), 150 mM NaCl, 1 mM DTT, 10 μM ZnSO4 containing 10% D2O, and 20 μM dimethylsilapentane-5-sulfonic acid. Spectra were acquired at 293 K on a Bruker DRX-600 spectrometer and were processed using Topspin (Bruker). A TBX20 pseudogene covering exons 5 and 6 exists on human chromosome 12. This shows 98.4% homology to cognate regions on chromosome 7. Exon 5 and 6 primers used for mutation screening were specific to TBX20 on chromosome 7. Coding-exon PCR primers are available on request. We screened for mutations in TBX20 coding exons by direct DNA sequencing in 352 probands with CHD, 175 with ASD, 63 with VSD, and 115 with some other form of cardiac structural anomaly (table 1). Probands were recruited without reference to family history of CHD. However, 39 individuals (11%) had at least one first-degree relative with CHD. CHD in most subjects was diagnosed during the newborn period or during early childhood, with 23% diagnosed during adulthood. The majority of subjects were white (76%); the remainder were Asian (including Indian and Pakistani, 11%), Pacific Islander (Polynesian and Melanesian, 6%), Middle Eastern (5%), or Australian Aboriginal (2%). Unique TBX20 mutations within exons encoding the T-box DNA-binding domain were found in two white probands with ASD, each with a positive family history of CHD. Family 1 carried the missense change TBX20 I152M (456C→G) (figs. 1, 2a, and 2b), which segregated with disease over 3 generations. The proband (III:1) had ASD, which was corrected surgically in early childhood. Her grandmother (I:2) had a small VSD, and her mother (II:2) had a large PFO with a permanent left-to-right blood shunt. Cardiac valves and left ventricular (LV) function were normal in all individuals. The I152M change was absent in >450 white controls.Figure 2Structural impact of TBX20 mutations illustrated with a model of the TBX20 T-box (blue ribbon) bound to DNA (gray surface) on the basis of the x-ray crystal structure of the TBX3 domain.23Coll M Seidman JG Muller CW Structure of the DNA-bound T-box domain of human TBX3, a transcription factor responsible for ulnar-mammary syndrome.Structure. 2002; 10: 343-356Abstract Full Text Full Text PDF PubMed Scopus (83) Google Scholara, Space-filling representation of affected residues showing Ile152 (yellow) located in the core of the T-box and Thr209 (green) at the DNA-interaction face. b, Side chain of Ile152, packed within the hydrophobic core. The extra length and possibility of additional rotation within the side chain of methionine may disrupt the hydrophobic packing in this region and destabilize the structure. c, Q195X, which results in truncation of the TBX20 protein within the T-box. The region of the T-box expressed in the Q195X variant is shown in blue, with the remainder of the domain