Gene 534 (2014) 320–323
Contents lists available at ScienceDirect
Gene journal homepage: www.elsevier.com/locate/gene
A novel mutation of GATA4 (K319E) is responsible for familial atrial septal defect and pulmonary valve stenosis Rong Xiang a,b,c, Liang-Liang Fan a, Hao Huang a, Bei-Bei Cao a, Xiang-Ping Li c, Dao-Quan Peng c,⁎, Kun Xia a,b,⁎⁎ a b c
Department of Cell Biology, School of Life Sciences, Central South University, Changsha 410013, China State Key Laboratory of Medical Genetics, Central South University, Changsha 410078, China Department of Cardiology, the Second Xiangya Hospital of Central South University, Changsha 410011, China
a r t i c l e
i n f o
Article history: Accepted 12 October 2013 Available online 27 October 2013 Keywords: Congenital heart disease Atrial septal defect ASD GATA4 Transcription factor
a b s t r a c t Congenital heart disease (CHD) is the most common birth defect in humans, and the etiology of most CHD remains to be elusive. Atrial septal defect (ASD) makes up 30–40% of all adult CHDs and is thought to be genetically heterogeneous. Previous studies have demonstrated that mutations in transcription factors e.g. NKX2.5, GATA4, and TBX5 contribute to congenital ASD. In this study, we investigate a family of three generations with seven patients with ASD and pulmonary valve stenosis (PS). A novel GATA4 mutation, c.955ANG (p.K319E), was identified and co-segregated with the affected patients in this family. This mutation was predicted to be deleterious by three different bioinformatics programs (The polyphen2, SIFT and MutationTaster). Our finding expands the spectrum of GATA4 mutations and provides additional support that GATA4 plays important roles in cardiac development. © 2013 Elsevier B.V. All rights reserved.
1. Introduction Congenital heart disease (CHD) is the most common birth defect and the leading non-infectious cause of mortality in newborns, affecting appropriately seven per 1000 of live births (Hoffman and Kaplan, 2002). It is estimated that about 130,000 new CHD cases increased in China annually (Zhao et al., 2013). Atrial septal defect (ASD) is defined by an anatomically deficient inter-atrial septum allowing oxygen-rich blood to flow directly from the left to the right atria. ASD makes up 30–40% of all adult CHD (Hoffman and Kaplan, 2002; Kaplan, 1993). Pulmonary valve stenosis has been reported in combination with ASD or as part of complex syndromes (e.g. Noonan syndrome) (Koretzky et al., 1969; Tartaglia et al., 2011). To date, more than ten genes of three groups underlying ASD have been identified. (i) Transcription factors and cofactors, e.g. GATA4, NKX2.5, GATA6, TBX5, ZIC3 and CITED2. (ii) Ligands–receptors, e.g. ALK2, CRELD1 and GJA1. (iii) Structure protein of sarcomere, e.g. MYH6, MYH7 and ACTC (Fahed et al., 2013; Wessels and Willems, 2010). However, due to significant genetic heterogeneity and scarcity of
Abbreviations: CHD, congenital heart defects; PS, pulmonary valve stenosis; polyphen2, polymorphism phenotyping; SIFT, Sorting Intolerant From Tolerant; dbSNP, Single Nucleotide Polymorphism Database; ASD, atrial septal defect; VSD, ventricular septal defect; TOF, tetralogy of Fallot; HRV, hypoplastic right ventricle; TAPVR, total anomalous pulmonary venous retour; NLS, nuclear localization signals; SNP, single nucleotide polymorphism; PCR, polymerase chain reaction. ⁎ Corresponding author. ⁎⁎ Correspondence to: K. Xia, Department of Cell Biology, School of Life Sciences, State Key Laboratory of Medical Genetics, Central South University, Changsha, China. E-mail addresses: [email protected] (D.-Q. Peng), [email protected] (K. Xia). 0378-1119/$ – see front matter © 2013 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.gene.2013.10.028
families with defined monogenic inheritance of isolated ASD, the etiology of most isolated ASD still remains to be elucidated. Over 20 mutations have been identified in previous mutation screenings of GATA4 with familial or sporadic CHD, while the causal interrelationships remain mostly speculative and long-term follow-up observations are needed to clarify the possible responsibility (Butler et al., 2010; Posch et al., 2010; Wang et al., 2013; Xiong et al., 2013; Zhang et al., 2008). In this study, we investigated the possible causative gene in a family with ASD and pulmonary valve stenosis (PS). We identified a de novo mutation (c.955ANG/p.K319E) in exon5 of GATA4 in all affected members of this family. To the best of our knowledge, this mutation has not been reported in previous study or presented in our control cohorts and dbSNP as well as Exome Variant Server database (http:// evs.gs.washington.edu/EVS/). 2. Material and methods The Review Board of the Second Xiangya Hospital of the Central South University has approved this research. All subjects have consented to this study. 2.1. Patients A family from Central-South China (Jiangxi Province) with 10 members across three generation participated in the present study. Seven patients were diagnosed of having ASD and PS (II2, II3, III1, III3, III4, III5 and IV1) (Fig. 1A, Table 1). The proband (III3) of the family was diagnosed by transthoracic echocardiograms of having ASD and PS with congestive heart failure. Patients' information is listed in
R. Xiang et al. / Gene 534 (2014) 320–323
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Fig. 1. (A). Pedigree of the family affected with ASD and PS. Family members are identified by generations and numbers. Squares indicate male family members; circles, female members; closed symbols, the affected members; open symbols, unaffected members; arrow, proband. (B). Sequencing results of the GATA4 mutation. Sequence chromatogram indicates an A to G transition of nucleotide 995.
Table 1. Family member IV1 was found to have ASD and PS just after birth and was surgically repaired at the age of four in the Department of Cardiothoracic Surgery of the Second Xiangya Hospital. No other
malformations were observed in the seven affected members and indicated this family to be an isolated or non-syndromic CHD family with autosomal dominant pattern.
322
R. Xiang et al. / Gene 534 (2014) 320–323
Table 1 Summary of a Chinese family with ASD and PS. ASD family member
III3 (proband) II1 II2 II3 II4 III1 III2 III4 III5 IV1
CHD
ASD, PS ASD, PS No ASD, PS No ASD, PS No ASD, PS ASD, PS ASD, PS
Age at diagnosis
32 y 57 y 59 y 56 y 56 y 35 y 36 y 34 y 30 y 4y
GATA4
Prediction by programs
DNA
Protein
Polyphen2
SIFT
MutationTaster
c.955ANG c.955ANG – c.955ANG – c.955ANG – c.955ANG c.955ANG c.955ANG
p.K319E p.K319E – p.K319E – p.K319E – p.K319E p.K319E p.K319E
Probably damaging (0.991)
Deleterious (0.02)
Disease-causing (1.53)
ASD, atrial septal defect; PS, pulmonary valve stenosis; y, year; SIFT, Sorting Intolerant From Tolerant.
2.2. Methods
4. Discussion
2.2.1. DNA extraction All family members gave written informed consent. Genomic DNA was prepared from peripheral blood of the patients and other all participants using a DNeasy Blood & Tissue Kit (Qiagen, Valencia, CA) on the QIAcube automated DNA extraction robot (Qiagen, Hilden, Germany).
GATA4 is a cardiac-specific member of the GATA family of zinc finger transcription factors characterized by their consensus DNA sequence ‘GATA’ motif (Huang et al., 1995). GATA4 contains two transcriptional activation domains (TAD1 and TAD2), two zinc finger domains (ZF1 and ZF2), and one nuclear localization signal (NLS). The C-terminal ZF1 is thought for DNA sequence recognition and binding to the consensus motif, while the N-terminal ZF2 is responsible for sequence specificity and stability of protein–DNA binding (Philips et al., 2007). In 2003, Mutations in GATA4 were identified in two families with highly penetrant ASD (Garg et al., 2003). Interestingly, a wide variety of congenital cardiovascular anomalies, including atrial septal defect (ASD) (Garg et al., 2003), ventricular septal defect (VSD) (TomitaMitchell et al., 2007), tetralogy of Fallot (TOF) (Zhang et al., 2008), pulmonary valve stenosis (PS) (Garg et al., 2003; Reamon-Buettner and Borlak, 2005), hypoplastic right ventricle (HRV) (Rajagopal et al., 2007) and total anomalous pulmonary venous retour (TAPVR) (Posch et al., 2008), have been reported in patients with GATA4 mutations. Similar observations were also reported in the families even with the same mutation (Garg et al., 2003). Therefore, the pleiotropic types of CHD and significant genetic heterogeneity make it difficult for the candidate gene approaches to find causative genes. In this study, a family with ASD and PS was investigated for mutation(s) in GATA4 NKX2–5 and TBX5. Genetic analysis revealed a GATA4 mutation (c.955AN G/p.K319E) in all seven affected patients with ASD, but not in any control individuals. Of note, all seven patients with p.K319E in this family were associated with PS. Similar findings have been reported in previous reports. To date, four mutations (T280M, G296S, M310V and S358fs) have been reported in ASD patients associated with PS (Chen et al., 2010a, 2010b; Garg et al., 2003; Misra et al., 2012; Okubo et al., 2004). Given that three out of four mutation sites are located in the second zinc finger motif and adjacent stretches of basic amino acids (aa 271–320, UniProtKB/Swiss-Prot entry P43694). It might be interesting to investigate the genotype–phenotype correlation between GATA4 mutations and pulmonary valve stenosis. Like all nuclear proteins, GATA4 has a nuclear localization signal region (NLS, aa 271–325 in humans) that allows GATA4 to be targeted to the nucleus via the nuclear pore complex (Ko and Engel, 1993; Philips et al., 2007). As demonstrated by mouse cell-line model, four amino acids Arg283, Arg284, Arg318, Arg320 (in humans) play crucial roles for nuclear localization of GATA4 (Philips et al., 2007). Very interestingly, the mutated site in our study, p.319K, just locates between the last two arginine residues (aa 318 and 320) (Fig. 2B). It is reasonable to speculate that this mutation may impair the interaction between importin-beta, GATA4 and DNA, though it remains to be further verified. In conclusion, we report a novel GATA4 mutation (p.K319E) in a three generation family with seven ASD and PS patients. The present identification of a novel mutation not only further supports
2.2.2. Mutation sequencing The entire coding regions, including the flanking intronic sequences of NKX2.5 (Refseq: NM_004387), GATA4 (NM_002052), TBX5 (NM_000192), were amplified with polymerase chain reaction (PCR; primer sequences will be provided upon requests). Sequences of the PCR products were determined using the ABI 3100 Genetic Analyzer (ABI, Foster City, CA) as previously described (Tan et al., 2012). 2.2.3. Multiple sequence alignments and bioinformatic prediction of mutation The multiple GATA4 protein sequences across mammals were aligned using the program MUSCLE (version 3.6, an online program at http://www.ncbi.nlm.nih.gov). The polyphen2 (polymorphism phenotyping, http://genetics.bwh. harvard.edu/pph2/) (Sunyaev et al., 2000), SIFT (Sorting Intolerant From Tolerant, http://sift.bii.astar.edu.sg/) (Ng and Henikoff, 2003) and MutationTaster (www.mutationtaster.org) (Schwarz et al., 2010) programs were used to predict the effects of these sequence variants on the function of the protein. 3. Results We describe a family with ASD and PS in which vertical transmission of the disease occurs, suggesting apparent autosomal dominant inheritance. We therefore examined the possibility of known causative genes that cause ASD. By sequencing analysis of NKX2.5, GATA4 and TBX5, a novel non-synonymous sequence variant, c.955AN G (p.K319E) in the exon5 of GATA4 was detected and co-segregated with the affected ASD family members (Fig. 1). This newly identified c.955ANG mutation was not found in our 200 control cohorts (Tan et al., 2012). This mutation was also not presented in dbSNP and Exome Variant Server database (http://evs.gs.washington.edu/EVS/). Alignment of GATA4 amino acid sequences from human, mouse, rat, dog, cow, chicken, zebrafish, etc., revealed that the affected amino acid was evolutionarily conserved (Fig. 2A). Three programs for analyzing protein functions, polyphen2, SIFT and MutationTaster, predicted that the p.K319E variants are probably damaging, deleterious and disease causing, respectively (Table 1). All three different algorithm based bioinformatics programs show a consistent result of detrimental effect of the variant, suggesting that the site (K319) plays important roles in the function of GATA4.
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Fig. 2. Analysis of the mutation and protein domains of GATA4. A. Alignment of multiple GATA4 protein sequences across species. The K319 affected amino acid locates in the highly conserved amino acid region in different mammals (from Ensembl). Blue column shows the K319 site. B. Schematic of GATA4 protein domains indicates N-terminal zinc finger (N-Znf), C-terminal zinc finger (C-Znf). A minimal nuclear localization signal (NLS) region (aa 271–325) is indicated in green line. The four critical amino acids for NLS (R283, R284, R318 and R320) are in blue characters, and the four mutated amino acids associated with PS are in red characters (T280, G296, M310 and K319).
the important role of cardiac transcription factor GATA4 in congenital ASD but also expands the spectrum of GATA4 mutations and will contribute to genetic diagnosis and counseling of families with CHD. Conflict of interest None. Acknowledgments We thank the patients and their families for participating in this study. We thank the State Key Laboratory of Medical Genetics of China for technical assistance. This study was supported by the National Basic Research Program of China (973 Program) (2012CB517900) and the National Natural Science Foundation of China (30800476). References Butler, T.L., et al., 2010. GATA4 mutations in 357 unrelated patients with congenital heart malformation. Genet. Test. Mol. Biomarkers 14, 797–802. Chen, Y., et al., 2010a. A novel mutation in GATA4 gene associated with dominant inherited familial atrial septal defect. J. Thorac. Cardiovasc. Surg. 140, 684–687. Chen, Y., et al., 2010b. A novel mutation of GATA4 in a familial atrial septal defect. Clin. Chim. Acta 411, 1741–1745. Fahed, A.C., Gelb, B.D., Seidman, J.G., Seidman, C.E., 2013. Genetics of congenital heart disease: the glass half empty. Circ. Res. 112, 707–720. Garg, V., et al., 2003. GATA4 mutations cause human congenital heart defects and reveal an interaction with TBX5. Nature 424, 443–447. Hoffman, J.I., Kaplan, S., 2002. The incidence of congenital heart disease. J. Am. Coll. Cardiol. 39, 1890–1900. Huang, W.Y., Cukerman, E., Liew, C.C., 1995. Identification of a GATA motif in the cardiac alpha-myosin heavy-chain-encoding gene and isolation of a human GATA-4 cDNA. Gene 155, 219–223. Kaplan, S., 1993. Congenital heart disease in adolescents and adults. Natural and postoperative history across age groups. Cardiol. Clin. 11, 543–556.
Ko, L.J., Engel, J.D., 1993. DNA-binding specificities of the GATA transcription factor family. Mol. Cell. Biol. 13, 4011–4022. Koretzky, E.D., Moller, J.H., Korns, M.E., Schwartz, C.J., Edwards, J.E., 1969. Congenital pulmonary stenosis resulting from dysplasia of valve. Circulation 40, 43–53. Misra, C., et al., 2012. Congenital heart disease-causing Gata4 mutation displays functional deficits in vivo. PLoS Genet. 8, e1002690. Ng, P.C., Henikoff, S., 2003. SIFT: predicting amino acid changes that affect protein function. Nucleic Acids Res. 31, 3812–3814. Okubo, A., et al., 2004. A novel GATA4 mutation completely segregated with atrial septal defect in a large Japanese family. J. Med. Genet. 41, e97. Philips, A.S., Kwok, J.C., Chong, B.H., 2007. Analysis of the signals and mechanisms mediating nuclear trafficking of GATA-4. Loss of DNA binding is associated with localization in intranuclear speckles. J. Biol. Chem. 282, 24915–24927. Posch, M.G., et al., 2008. Mutations in GATA4, NKX2.5, CRELD1, and BMP4 are infrequently found in patients with congenital cardiac septal defects. Am. J. Med. Genet. A 146A, 251–253. Posch, M.G., Perrot, A., Berger, F., Ozcelik, C., 2010. Molecular genetics of congenital atrial septal defects. Clin. Res. Cardiol. 99, 137–147. Rajagopal, S.K., et al., 2007. Spectrum of heart disease associated with murine and human GATA4 mutation. J. Mol. Cell. Cardiol. 43, 677–685. Reamon-Buettner, S.M., Borlak, J., 2005. GATA4 zinc finger mutations as a molecular rationale for septation defects of the human heart. J. Med. Genet. 42, e32. Schwarz, J.M., Rodelsperger, C., Schuelke, M., Seelow, D., 2010. MutationTaster evaluates disease-causing potential of sequence alterations. Nat. Methods 7, 575–576. Sunyaev, S., Ramensky, V., Bork, P., 2000. Towards a structural basis of human nonsynonymous single nucleotide polymorphisms. Trends Genet. 16, 198–200. Tan, Z.P., Huang, C., Xu, Z.B., Yang, J.F., Yang, Y.F., 2012. Novel ZFPM2/FOG2 variants in patients with double outlet right ventricle. Clin. Genet. 82, 466–471. Tartaglia, M., Gelb, B.D., Zenker, M., 2011. Noonan syndrome and clinically related disorders. Best Pract. Res. Clin. Endocrinol. Metab. 25, 161–179. Tomita-Mitchell, A., Maslen, C.L., Morris, C.D., Garg, V., Goldmuntz, E., 2007. GATA4 sequence variants in patients with congenital heart disease. J. Med. Genet. 44, 779–783. Wang, E., et al., 2013. Identification of functional mutations in GATA4 in patients with congenital heart disease. PLoS One 8, e62138. Wessels, M.W., Willems, P.J., 2010. Genetic factors in non-syndromic congenital heart malformations. Clin. Genet. 78, 103–123. Xiong, F., et al., 2013. Analyses of GATA4, NKX2.5, and TFAP2B genes in subjects from southern China with sporadic congenital heart disease. Cardiovasc. Pathol. 22, 141–145. Zhang, W., Li, X., Shen, A., Jiao, W., Guan, X., Li, Z., 2008. GATA4 mutations in 486 Chinese patients with congenital heart disease. Eur. J. Med. Genet. 51, 527–535. Zhao, Q.M., Ma, X.J., Jia, B., Huang, G.Y., 2013. Prevalence of congenital heart disease at live birth: an accurate assessment by echocardiographic screening. Acta Paediatr. 102, 397–402.
Contents lists available at ScienceDirect
Gene journal homepage: www.elsevier.com/locate/gene
A novel mutation of GATA4 (K319E) is responsible for familial atrial septal defect and pulmonary valve stenosis Rong Xiang a,b,c, Liang-Liang Fan a, Hao Huang a, Bei-Bei Cao a, Xiang-Ping Li c, Dao-Quan Peng c,⁎, Kun Xia a,b,⁎⁎ a b c
Department of Cell Biology, School of Life Sciences, Central South University, Changsha 410013, China State Key Laboratory of Medical Genetics, Central South University, Changsha 410078, China Department of Cardiology, the Second Xiangya Hospital of Central South University, Changsha 410011, China
a r t i c l e
i n f o
Article history: Accepted 12 October 2013 Available online 27 October 2013 Keywords: Congenital heart disease Atrial septal defect ASD GATA4 Transcription factor
a b s t r a c t Congenital heart disease (CHD) is the most common birth defect in humans, and the etiology of most CHD remains to be elusive. Atrial septal defect (ASD) makes up 30–40% of all adult CHDs and is thought to be genetically heterogeneous. Previous studies have demonstrated that mutations in transcription factors e.g. NKX2.5, GATA4, and TBX5 contribute to congenital ASD. In this study, we investigate a family of three generations with seven patients with ASD and pulmonary valve stenosis (PS). A novel GATA4 mutation, c.955ANG (p.K319E), was identified and co-segregated with the affected patients in this family. This mutation was predicted to be deleterious by three different bioinformatics programs (The polyphen2, SIFT and MutationTaster). Our finding expands the spectrum of GATA4 mutations and provides additional support that GATA4 plays important roles in cardiac development. © 2013 Elsevier B.V. All rights reserved.
1. Introduction Congenital heart disease (CHD) is the most common birth defect and the leading non-infectious cause of mortality in newborns, affecting appropriately seven per 1000 of live births (Hoffman and Kaplan, 2002). It is estimated that about 130,000 new CHD cases increased in China annually (Zhao et al., 2013). Atrial septal defect (ASD) is defined by an anatomically deficient inter-atrial septum allowing oxygen-rich blood to flow directly from the left to the right atria. ASD makes up 30–40% of all adult CHD (Hoffman and Kaplan, 2002; Kaplan, 1993). Pulmonary valve stenosis has been reported in combination with ASD or as part of complex syndromes (e.g. Noonan syndrome) (Koretzky et al., 1969; Tartaglia et al., 2011). To date, more than ten genes of three groups underlying ASD have been identified. (i) Transcription factors and cofactors, e.g. GATA4, NKX2.5, GATA6, TBX5, ZIC3 and CITED2. (ii) Ligands–receptors, e.g. ALK2, CRELD1 and GJA1. (iii) Structure protein of sarcomere, e.g. MYH6, MYH7 and ACTC (Fahed et al., 2013; Wessels and Willems, 2010). However, due to significant genetic heterogeneity and scarcity of
Abbreviations: CHD, congenital heart defects; PS, pulmonary valve stenosis; polyphen2, polymorphism phenotyping; SIFT, Sorting Intolerant From Tolerant; dbSNP, Single Nucleotide Polymorphism Database; ASD, atrial septal defect; VSD, ventricular septal defect; TOF, tetralogy of Fallot; HRV, hypoplastic right ventricle; TAPVR, total anomalous pulmonary venous retour; NLS, nuclear localization signals; SNP, single nucleotide polymorphism; PCR, polymerase chain reaction. ⁎ Corresponding author. ⁎⁎ Correspondence to: K. Xia, Department of Cell Biology, School of Life Sciences, State Key Laboratory of Medical Genetics, Central South University, Changsha, China. E-mail addresses: [email protected] (D.-Q. Peng), [email protected] (K. Xia). 0378-1119/$ – see front matter © 2013 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.gene.2013.10.028
families with defined monogenic inheritance of isolated ASD, the etiology of most isolated ASD still remains to be elucidated. Over 20 mutations have been identified in previous mutation screenings of GATA4 with familial or sporadic CHD, while the causal interrelationships remain mostly speculative and long-term follow-up observations are needed to clarify the possible responsibility (Butler et al., 2010; Posch et al., 2010; Wang et al., 2013; Xiong et al., 2013; Zhang et al., 2008). In this study, we investigated the possible causative gene in a family with ASD and pulmonary valve stenosis (PS). We identified a de novo mutation (c.955ANG/p.K319E) in exon5 of GATA4 in all affected members of this family. To the best of our knowledge, this mutation has not been reported in previous study or presented in our control cohorts and dbSNP as well as Exome Variant Server database (http:// evs.gs.washington.edu/EVS/). 2. Material and methods The Review Board of the Second Xiangya Hospital of the Central South University has approved this research. All subjects have consented to this study. 2.1. Patients A family from Central-South China (Jiangxi Province) with 10 members across three generation participated in the present study. Seven patients were diagnosed of having ASD and PS (II2, II3, III1, III3, III4, III5 and IV1) (Fig. 1A, Table 1). The proband (III3) of the family was diagnosed by transthoracic echocardiograms of having ASD and PS with congestive heart failure. Patients' information is listed in
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Fig. 1. (A). Pedigree of the family affected with ASD and PS. Family members are identified by generations and numbers. Squares indicate male family members; circles, female members; closed symbols, the affected members; open symbols, unaffected members; arrow, proband. (B). Sequencing results of the GATA4 mutation. Sequence chromatogram indicates an A to G transition of nucleotide 995.
Table 1. Family member IV1 was found to have ASD and PS just after birth and was surgically repaired at the age of four in the Department of Cardiothoracic Surgery of the Second Xiangya Hospital. No other
malformations were observed in the seven affected members and indicated this family to be an isolated or non-syndromic CHD family with autosomal dominant pattern.
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Table 1 Summary of a Chinese family with ASD and PS. ASD family member
III3 (proband) II1 II2 II3 II4 III1 III2 III4 III5 IV1
CHD
ASD, PS ASD, PS No ASD, PS No ASD, PS No ASD, PS ASD, PS ASD, PS
Age at diagnosis
32 y 57 y 59 y 56 y 56 y 35 y 36 y 34 y 30 y 4y
GATA4
Prediction by programs
DNA
Protein
Polyphen2
SIFT
MutationTaster
c.955ANG c.955ANG – c.955ANG – c.955ANG – c.955ANG c.955ANG c.955ANG
p.K319E p.K319E – p.K319E – p.K319E – p.K319E p.K319E p.K319E
Probably damaging (0.991)
Deleterious (0.02)
Disease-causing (1.53)
ASD, atrial septal defect; PS, pulmonary valve stenosis; y, year; SIFT, Sorting Intolerant From Tolerant.
2.2. Methods
4. Discussion
2.2.1. DNA extraction All family members gave written informed consent. Genomic DNA was prepared from peripheral blood of the patients and other all participants using a DNeasy Blood & Tissue Kit (Qiagen, Valencia, CA) on the QIAcube automated DNA extraction robot (Qiagen, Hilden, Germany).
GATA4 is a cardiac-specific member of the GATA family of zinc finger transcription factors characterized by their consensus DNA sequence ‘GATA’ motif (Huang et al., 1995). GATA4 contains two transcriptional activation domains (TAD1 and TAD2), two zinc finger domains (ZF1 and ZF2), and one nuclear localization signal (NLS). The C-terminal ZF1 is thought for DNA sequence recognition and binding to the consensus motif, while the N-terminal ZF2 is responsible for sequence specificity and stability of protein–DNA binding (Philips et al., 2007). In 2003, Mutations in GATA4 were identified in two families with highly penetrant ASD (Garg et al., 2003). Interestingly, a wide variety of congenital cardiovascular anomalies, including atrial septal defect (ASD) (Garg et al., 2003), ventricular septal defect (VSD) (TomitaMitchell et al., 2007), tetralogy of Fallot (TOF) (Zhang et al., 2008), pulmonary valve stenosis (PS) (Garg et al., 2003; Reamon-Buettner and Borlak, 2005), hypoplastic right ventricle (HRV) (Rajagopal et al., 2007) and total anomalous pulmonary venous retour (TAPVR) (Posch et al., 2008), have been reported in patients with GATA4 mutations. Similar observations were also reported in the families even with the same mutation (Garg et al., 2003). Therefore, the pleiotropic types of CHD and significant genetic heterogeneity make it difficult for the candidate gene approaches to find causative genes. In this study, a family with ASD and PS was investigated for mutation(s) in GATA4 NKX2–5 and TBX5. Genetic analysis revealed a GATA4 mutation (c.955AN G/p.K319E) in all seven affected patients with ASD, but not in any control individuals. Of note, all seven patients with p.K319E in this family were associated with PS. Similar findings have been reported in previous reports. To date, four mutations (T280M, G296S, M310V and S358fs) have been reported in ASD patients associated with PS (Chen et al., 2010a, 2010b; Garg et al., 2003; Misra et al., 2012; Okubo et al., 2004). Given that three out of four mutation sites are located in the second zinc finger motif and adjacent stretches of basic amino acids (aa 271–320, UniProtKB/Swiss-Prot entry P43694). It might be interesting to investigate the genotype–phenotype correlation between GATA4 mutations and pulmonary valve stenosis. Like all nuclear proteins, GATA4 has a nuclear localization signal region (NLS, aa 271–325 in humans) that allows GATA4 to be targeted to the nucleus via the nuclear pore complex (Ko and Engel, 1993; Philips et al., 2007). As demonstrated by mouse cell-line model, four amino acids Arg283, Arg284, Arg318, Arg320 (in humans) play crucial roles for nuclear localization of GATA4 (Philips et al., 2007). Very interestingly, the mutated site in our study, p.319K, just locates between the last two arginine residues (aa 318 and 320) (Fig. 2B). It is reasonable to speculate that this mutation may impair the interaction between importin-beta, GATA4 and DNA, though it remains to be further verified. In conclusion, we report a novel GATA4 mutation (p.K319E) in a three generation family with seven ASD and PS patients. The present identification of a novel mutation not only further supports
2.2.2. Mutation sequencing The entire coding regions, including the flanking intronic sequences of NKX2.5 (Refseq: NM_004387), GATA4 (NM_002052), TBX5 (NM_000192), were amplified with polymerase chain reaction (PCR; primer sequences will be provided upon requests). Sequences of the PCR products were determined using the ABI 3100 Genetic Analyzer (ABI, Foster City, CA) as previously described (Tan et al., 2012). 2.2.3. Multiple sequence alignments and bioinformatic prediction of mutation The multiple GATA4 protein sequences across mammals were aligned using the program MUSCLE (version 3.6, an online program at http://www.ncbi.nlm.nih.gov). The polyphen2 (polymorphism phenotyping, http://genetics.bwh. harvard.edu/pph2/) (Sunyaev et al., 2000), SIFT (Sorting Intolerant From Tolerant, http://sift.bii.astar.edu.sg/) (Ng and Henikoff, 2003) and MutationTaster (www.mutationtaster.org) (Schwarz et al., 2010) programs were used to predict the effects of these sequence variants on the function of the protein. 3. Results We describe a family with ASD and PS in which vertical transmission of the disease occurs, suggesting apparent autosomal dominant inheritance. We therefore examined the possibility of known causative genes that cause ASD. By sequencing analysis of NKX2.5, GATA4 and TBX5, a novel non-synonymous sequence variant, c.955AN G (p.K319E) in the exon5 of GATA4 was detected and co-segregated with the affected ASD family members (Fig. 1). This newly identified c.955ANG mutation was not found in our 200 control cohorts (Tan et al., 2012). This mutation was also not presented in dbSNP and Exome Variant Server database (http://evs.gs.washington.edu/EVS/). Alignment of GATA4 amino acid sequences from human, mouse, rat, dog, cow, chicken, zebrafish, etc., revealed that the affected amino acid was evolutionarily conserved (Fig. 2A). Three programs for analyzing protein functions, polyphen2, SIFT and MutationTaster, predicted that the p.K319E variants are probably damaging, deleterious and disease causing, respectively (Table 1). All three different algorithm based bioinformatics programs show a consistent result of detrimental effect of the variant, suggesting that the site (K319) plays important roles in the function of GATA4.
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Fig. 2. Analysis of the mutation and protein domains of GATA4. A. Alignment of multiple GATA4 protein sequences across species. The K319 affected amino acid locates in the highly conserved amino acid region in different mammals (from Ensembl). Blue column shows the K319 site. B. Schematic of GATA4 protein domains indicates N-terminal zinc finger (N-Znf), C-terminal zinc finger (C-Znf). A minimal nuclear localization signal (NLS) region (aa 271–325) is indicated in green line. The four critical amino acids for NLS (R283, R284, R318 and R320) are in blue characters, and the four mutated amino acids associated with PS are in red characters (T280, G296, M310 and K319).
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