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1、Functional analysis of two novel mutations in TWIST1 protein motifs found in ventricular septal defect patients DENG Xiaopeng1, WANG Binbin2,3, WANG Jing2,3, PAN Hong2,3, CHENG Zhi2,3,5CHENG Longfei2,3, ZHAO Lixi2,3, LI Hui1, MA Xu2,3,4(1. Departmen t of Obstetrics and Gynecology, Shengjing Hospital
2、 of China Medical University,ShenYang 110004;2. Graduate school, Peking Union Medical College, Beijing 100081;3. National Research Institute for Family Planning, Beijing 100081;104. World Health Organization Collaborating Centre for Research in Human Reproduction,Beijing 100081)Abstract: Aims: To in
3、vestigate the possible genetic effect of sequence variations in TWIST1 on the pathogenesis of ventricular septal defect in humans. Methods: We examined the coding region of TWIST1 in a cohort of 196 Chinese people with non-syndromic ventricular septal defect patients and15200 healthy individuals as
4、the controls.Results: We identified two novel potential disease-associated mutations, NM_000474.3:c.247GA (p.Gly83Ser) and NM_000474.3:c.283AG (p. Ser95Gly). Bothof them were identified for the first time and were not observed in the 200 controls without congenital heart disease. Using a dual-lucife
5、rase reporter assay, we showed that both of the mutations significantly down-regulated the repressive effect of TWIST1 on the E-cadherin promoter. Furthermore, a20mammalian two-hybrid assay showed that both of the mutations significantly affected the interaction between TWIST1 and KAT2B. Conclusions
6、: New mutations in the transcription factor TWIST1 that affect protein function were identified in 1.0% (2/196) of Chinese patients with ventricular septal defect. Our data show, for the first time, that TWIST1 has a potential causative effect on thedevelopment of ventricular septal defect.25Keyword
7、s: TWIST1; ventricular septal defect; variant; genetics0IntroductionFormation of the heart is one of the most complex processes during mammalian development. This formation is dependent on well-harmonized interplay and regulation of a variety of molecular30and morphogenetic events (1). The complexit
8、y of its development renders the heart vulnerable to congenital diseases, affecting approximately 12% of all live births and is the leading cause ofdeath in infants under 1 year old（2）.Over the past couple decades, a variety of genes, mainly encoding transcription factors, which are related to both
9、hereditary and sporadic congenital heart disease (CHD), have been35identified (3). Moreover, a series of transcription factors that affect the processes of controlling morphogenesis of the developing heart have been extensively studied. In recent years, a variety of heterozygous mutations in several
10、 transcription factors, such as NOTCH1(4), NKX2.5(5), and GATA4(6), have been identified to be involved in the etiology of CHD and have been recently reviewed(7).40The Twist-family of basic helix-loop-helix factors is a highly evolutionarily conserved family of proteins in most multicellular organis
11、ms, and they play essential roles in both embryological morphogenesis and pathological disease(8-10). The TWIST1 gene, also known as TWIST, is located on human chromosome 7p21, it comprises 2 exons, and encodes a 202 amino acid protein. Previous studies had shown that TWIST1 mutations are involved i
12、n the Saether-Chotzen45syndrome, a haploinsufficient disorder, which primarily affects cranial and limb development (11).Foundations: 教育部高等学校博士学科点专项科研基金（20092104110011）；辽宁省科学技术计划（2011225017）Brief author introduction:邓晓鹏（1982-），女，医师，胎儿产前诊断Correspondance author: Li Hui（1970-),Woman，Prof.，Department of
13、 Obstetrics and Gynecology,. E-mail:FCKLIHUIRecently, approximately 70 mutant alleles of TWIST1 have been found in patients with Saether-Chotzen syndrome (12). The deduced protein of TWIST1 has a hydrophilic N terminus, followed by a repeat region that encodes the glycine-rich sequence (Glycine)5Ala
14、nine(Glycine)5 and a conserved basic-helix-loop-helix motif in the C-terminal half (13,14). The TWIST1 gene50was initially identified in Drosophila in which it was implicated in mesodermal patterning duringmultiple steps of early embryonic development(15). In the vertebrate, TWIST1 is necessary for
15、gastrulation and is expressed in the prospective mesoderm(9,16). In addition, within the developing mammalian heart, TWIST1 is present in the endocardial cushions of the atrioventricular canal and outflow tract, in which it has been proposed to induce55epithelial-mesenchymal transitions (17,18). TWI
16、ST1 in the endocardial cushions is subject to BMP2 regulation and induces Tbx20 expression(18-20). TWIST1 gain and loss of function experiments were performed in primary chicken and mice endocardial cushion cells to determine its role in endocardial cushion development (10,18). These studies indicat
17、ed that TWIST1 induces endocardial cushion cell proliferation and promotes endocardial cushion cell migration. Moreover,60TWIST1 deletion in the mouse impairs cardiac neural crest migration and exhibits cardiac outflow tract defects (10).We hypothesize that TWIST1 contributes to the development of C
18、HD. The present studyaimed to examine the etiology of CHD by identifying potential pathogenic mutations in theTWIST1 gene in Chinese ventricular septal defect (VSD) patients and to provide important insight65into the etiology of CHD.1Materials and Methods1.1Study populationA total of 196 Chinese VSD
19、 patients and 200 healthy control subjects with no reported cardiac phenotypes were recruited for this study from Lanzhou University. Informed consent was70obtained from patients parents or guardians. The study protocol conformed to the ethicalguidelines of the 1975 Declaration of Helsinki and was a
20、pproved by the Ethics Committee of theNational Research Institute for Family Planning.Clinical assessment of the patients included anthropometric measurements, a physical examination for dysmorphism and malformation, and radiological evaluation. The patients also75underwent a chest X-ray examination
21、, electrocardiogram and ultrasonic echocardiogram.1.2Mutational and bioinformatics analysesGenomic DNA was extracted from human peripheral blood leukocytes using a standard method. The human TWIST1 gene is located on 7p21, it has two exons, and is encoded by the first exon. The first exon and flanki
22、ng introns of TWIST1 were amplified by polymerase chain reaction80(PCR) using one pair of TWIST1 gene-specific primers (supplementary Table 1). PCR products were sequenced using the appropriate PCR primers and the BigDye Terminator Cycle Sequencing Kit (Applied Biosystems, USA) and run on an automat
23、ed sequencer, the ABI 3730XL (Applied Biosystems), to perform mutational analysis.85Supplementary Table 1.TWIST1 gene primer and sizeExon Primer Sequence (5 to 3) Size (bp)1 TWIST1-1F GAGGCGCCCCGCTCTTCTCCTWIST1-1RCGGTTCCCAGTCCACCTCGATT817The novelty of all variants found in sequencing was first dete
24、rmined from the National9095100Centre for Biotechnology Information (NCBI) human SNP database (dbSNP) and the 1000Genome Project database (http:/browser.1000genomes.org/). The amino acid sequences of other species were obtained from NCBI Genebank, and conservation analysis was performed by CLC Main
25、Workbench Software (Aarhus, Denmark).1.3Site-directed mutagenesis and plasmid constructionHuman TWIST1 cDNA and KAT2B cDNA were obtained from OriGene True-Clone (Origene, USA). The pcDNA3.1 vector and the pGL3-basic vector were saved in our laboratory. The TK promoter pGL3-basic vector with four GAL
26、4 DNA-binding sites, the pCMX-GAL4 vector containing GAL4-DBD, and the pCMX-VP16 vector with the potent trans-activating domain of HSV VP16 were provided by Dr. Ronald M. Evans (The Salk Institute for Biological Studies, USA).Site-directed mutagenesis was performed using a QuickChange Site-Directed
27、Mutagenesis kit (Stratagene, USA) to generate human TWIST1 mutants bearing p.Gly83Ser or p.Ser95Gly by PCR-mediated mutagenesis with appropriate primers (supplementary Table 2). DNA sequencing confirmed the produced mutation.Supplementary Table2.Site-directed mutagenesis primerPrimer Name Primer Seq
28、uence (5 to 3)g247a5-gggctgtggcagcggcggcgg-3 g247a_antisense 5-ccgccgccgctgccacagccc-3 a283g5-cggcagcagcggcggcggcgg-3a283g_antisense 5-ccgccgccgccgctgctgccg-3105110115The open reading frames of wild-type and mutant TWIST1 were amplified by PCR from cDNA and cloned into a BamHI and EcoRI double diges
29、ted pcDNA3.1 vector, generating pcDNA3.1-TWIST1 vectors. The promoter region of human E-cadherin was amplified by PCR from human normal genomic DNA and inserted into an XhoI and KpnI double digested pGL3-basic vector, to create a human pGL3-E-cadherin promoter reporter plasmid. To create GAL4-TWIST1
30、 fusion protein, the opening frames of wild-type and mutant TWIST1 were amplified by PCR and cloned into an EcoRI and BamHI double digested pCMX-GAL4 vector. The opening reading frames of KAT2B were amplified by PCR from human KAT2B cDNA and inserted into a NheI single digested pCMX-VP16 vector to c
31、reate the VP16-KAT2B fusion protein. All the clones were confirmed by sequencing. The nucleotide sequences of the PCR primers used above are shown in Supplementary Table3.Supplementary Table3.Vectors and primersVectorpcDNA3.1- TWIST1 pGL3-basic-E-cadherin-promoterpCMX-VP16-KAT2BPrimer SequenceEnzyme
32、 siteSense CGCGGATCC gagAtgatgcaggacgtgtc BamH IAntisense CCGGAATTC ctagtgggacgcggaca EcoR I SenseGGGGTACC AGCCTGGCCAACATGGTGA KpnIAntisense CCGCTCGAG TGCAGTTCCGACGCCACT XhoI SenseCTAGCTAGC aaacaagaagatcctgatgtggc Nhe I AntisenseCTAGCTAGC GCTACTGTGTGCAATTTCCTCA Nhe IpCMX -GAL4-TWIST1Sense CCGGAATTCg
33、agCtgatgcaggacgtgtc EcoR IAntisense CGCGGATCCctagtgggacgcggaca BamH I1201.4Cell culture and transient transfectionThe 293T cells were maintained in Iscoves modified Dulbeccos medium supplemented with10% fetal bovine serum, 100 mg/ml penicillin and 100 mg/ml streptomycin in a humidified atmosphere co
34、ntaining 5% CO2 at 37C. Transfection was carried out using a standard calcium phosphate method or Lipofectamine 2000 (Invitrogen Corporation, Carlsbad, CA, USA).1251301351401.5Dual-luciferase reporter assayCultured 293T cells were co-transfected with pcDNA3.1-TWIST1 (wild-type or mutant), human pGL3
35、-E-cadherin promoter reporter plasmid and the Renilla luciferase control plasmid pREP7-RLu. Expression of Renilla luciferase provides an internal control to monitor the transfection efficiency. Forty-eight hours after transfection, the cells were gently rinsed with phosphate-buffered saline and harv
36、ested with Passive Lysis Buffer (Promega, USA). The Dual-luciferase Reporter Assay System (Promega, USA) was used to measure luciferase activity according to the manufacturers instructions. The Students t-test was used to determine statistical significance of unpaired samples.1.6Mammalian two-hybrid
37、 assayThe 293T cells were co-transfected with mammalian two-hybrid assay plasmids consisting of pCMX-GAL4-TWIST1 (wild-type or mutant) or pCMX-VP16-KAT2B, the TK promoter reporter plasmid, and the Renilla luciferase control plasmid pREP7-RLu. Thirty hours post-transfection, cells were washed and lys
38、ed in passive lysis buffer (Promega, USA) and the transfection efficiency was normalized to paired Renilla luciferase activity by using the Dual Luciferase Reporter Assay System (Promega, USA) according to the manufacturers instructions. The Students t-test was used to determine statistical signific
39、ance of unpaired samples.2Results1451501552.1Genetic and bioinformatics analysesIn our genetic analysis of transcription factor TWIST1 in 196 Chinese non-syndromic VSDpatients, we identified two novel non-synonymous variants in two individuals (2/196, 1%). A5-year-old male patient showed a substitut
40、ion of NM_000474.3:c.247GA (p.Gly83Ser) and theNM_000474.3:c.283AG (p.Ser95Gly) variant was found in a 7-month-old male patient (Figure1A). These variants were not found in the 200 normal controls without CHDs, suggesting that these variants are potentially involved in the etiology of VSD.Both poten
41、tial pathogenic variants have not been previously reported in the NCBI dbSNP and are not contained in the 1000 Genome Project database (http:/browser.1000genomes.org/).Both novel variants were located at the hydrophilic N terminus, which is the domain where TWIST1 interacts with KAT2B. Moreover, the
42、 variant NM_000474.3:c.247GA (p.Gly83Ser) was located at the glycine-rich sequence (Glycine)5Alanine(Glycine)5 (Figure 1B). Glycine83 andSerine95 are located at highly conserved regions among several species (Figure 1C).We used the online tool ExPASy (http:/www.expasy.org/cgi-bin/protscale.pl). This
43、 tool is defined by a numerical value assigned to each type of amino acid to predict the hydrophobicity or hydrophilicity and the secondary structure conformational parameters, as well as many other parameters, which are based on different chemical and physical properties of amino acids. Thehydropat
44、hicity of both mutant proteins was different from that of the wild type (Figure 1D).160165(A)Mutant 1c. G247A170ControlMutant 2c.A283G175180185Control(B)190(C)(D)(E)195200Figure 1. (A) Mutation analysis of the TWIST1 gene. The sequence chromatogram (reverse strand) shows two heterozygous transitions
45、, CT and TC, which resulted in the substitution of glycine by serine at codon 83 and serine by glycine at codon 95, respectively. The black arrows show the wild-type (unaffected individuals and the control) and mutation points (patients). (B) Schematic representation of the structure of the human TW
46、IST1 protein and positions of variants in the TWIST1 protein identified in VSD patients. (C) A Sequence alignment of TWIST1 proteins among several species. (D) Difference between the hydrophilicity of the wild type protein and the G83S mutant protein when predicted by ExPASy. (E) Difference between the hydro
