CASZ1 loss-of-function mutation associated with congenital heart disease

Gene 595 (2016) 62–68

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Research paper

CASZ1 loss-of-function mutation associated with congenital heart disease Ri-Tai Huang a, Song Xue a, Juan Wang b, Jian-Yun Gu c, Jia-Hong Xu c, Yan-Jie Li d, Ning Li d, Xiao-Xiao Yang d, Hua Liu d, Xiao-Dong Zhang d, Xin-Kai Qu d, Ying-Jia Xu d, Xing-Biao Qiu d, Ruo-Gu Li d,⁎, Yi-Qing Yang d,e,f,⁎ a

Department of Cardiovascular Surgery, Renji Hospital, School of Medicine, Shanghai Jiao Tong University, 1630 Dongfang Road, Shanghai 200127, PR China Department of Cardiovascular Medicine, East Hospital, Tongji University School of Medicine, 150 Jimo Road, Shanghai 200120, PR China Department of Cardiology, Tongji Hospital, Tongji University School of Medicine, 389 Xincun Road, Shanghai 200065, PR China d Department of Cardiology, Shanghai Chest Hospital, Shanghai Jiao Tong University, 241 West Huaihai Road, Shanghai 200030, PR China e Department of Cardiovascular Research Laboratory, Shanghai Chest Hospital, Shanghai Jiao Tong University, 241 West Huaihai Road, Shanghai 200030, PR China f Department of Central Laboratory, Shanghai Chest Hospital, Shanghai Jiao Tong University, 241 West Huaihai Road, Shanghai 200030, PR China b c

a r t i c l e

i n f o

Article history: Received 18 August 2016 Received in revised form 21 September 2016 Accepted 27 September 2016 Available online 28 September 2016 Keywords: Congenital heart disease Genetics Transcription factor CASZ1 Reporter gene assay

a b s t r a c t As the most common form of birth defect in humans, congenital heart disease (CHD) is associated with substantial morbidity and mortality in both children and adults. Increasing evidence demonstrates that genetic defects play a pivotal role in the pathogenesis of CHD. However, CHD is of great heterogeneity, and in an overwhelming majority of cases, the genetic determinants underpinning CHD remain elusive. In the present investigation, the coding exons and flanking introns of the CASZ1 gene, which codes for a zinc finger transcription factor essential for the cardiovascular morphogenesis, were sequenced in 172 unrelated patients with CHD. As a result, a novel heterozygous CASZ1 mutation, p.L38P, was identified in an index patient with congenital ventricular septal defect (VSD). Genetic scanning of the mutation carrier's available family members revealed that the mutation was present in all affected patients but absent in unaffected individuals. Analysis of the proband's pedigree showed that the mutation co-segregated with VSD, which was transmitted as an autosomal dominant trait with complete penetrance. The missense mutation, which altered the amino acid that was highly conserved evolutionarily, was absent in 200 unrelated, ethnically-matched healthy subjects used as controls. Functional deciphers by using a dual-luciferase reporter assay system unveiled that the mutant CASZ1 had significantly reduced transcriptional activity as compared with its wild-type counterpart. To the best of our knowledge, the current study firstly identifies CASZ1 as a new gene predisposing to CHD in humans, which provides novel insight into the molecular mechanisms underlying CHD and a potential therapeutic target for CASZ1-associated CHD, suggesting potential implications for personalized prophylaxis and therapy of CHD. © 2016 Published by Elsevier B.V.

1. Introduction Congenital heart disease (CHD), also known as congenital cardiovascular defect, is the most common form of birth defect in humans, accounting for approximately one-third of all major congenital abnormalities (Postma et al., 2016). Worldwide, 1.35 million infants are born with CHD each year, with an estimated overall incidence of 1 per 100 total births (Helbing et al., 2011; Mozaffarian et al., 2015; Postma et al., 2016). Now, CHD is still the most common cause of infant

Abbreviations: CHD, congenital heart disease; VSD, ventricular septal defect; PCR, polymerase chain reaction; TH-luc, tyrosine hydroxylase- luciferase; HEK, human embryonic kidney. ⁎ Corresponding authors at: Department of Cardiology, Shanghai Chest Hospital, Shanghai Jiao Tong University, 241 West Huaihai Road, Shanghai, 200030, PR China. E-mail addresses: [email protected] (R.-G. Li), [email protected] (Y.-Q. Yang).

http://dx.doi.org/10.1016/j.gene.2016.09.044 0378-1119/© 2016 Published by Elsevier B.V.

demise resulting from birth defects, with about 24% of infants who died of a birth defect having a cardiac developmental anomaly (Mozaffarian et al., 2015). As a structural problem attributed to abnormal development of the heart or major cardiothoracic blood vessel, CHD is usually categorized into 25 diverse clinical types, of which 21 designate specific anatomic or hemodynamic lesions, including ventricular septal defect (VSD), atrial septal defect, tetralogy of Fallot, double outlet right ventricle, truncus arteriosus, patent ductus arteriosus, endocardial cushion defect, transposition of the great arteries, aortic stenosis, coarctation of the aorta, pulmonary stenosis, pulmonary atresia and abnormal pulmonary venous connection (Mozaffarian et al., 2015). Although minor cardiovascular defects may resolve spontaneously (Mozaffarian et al., 2015), major malformations can necessitate timely surgical procedures and may result in degraded quality of life (Kahr et al., 2015) or depression (Diller et al., 2016), diminished exercise performance (Rosenblum et al., 2015), retarded cerebral development (Peyvandi et al., 2016) or

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brain injury (Marelli et al., 2016), cerebral or pulmonary thrombosis (Jensen et al., 2015), infective endocarditis (Rushani et al., 2013), pulmonary arterial hypertension or Eisenmenger's syndrome (Dimopoulos et al., 2014), cardiac liver cirrhosis (Ford et al., 2015), impaired renal function (Zheng et al., 2013), chronic heart failure (Stout et al., 2016), arrhythmias (McLeod and Warnes, 2016) and even sudden unexpected death (Jortveit et al., 2016). Although enormous advance in pediatric cardiovascular care has allowed over 85% of newborns with CHD to survive into adulthood, it gives rise to an increasing number of adults living with CHD, and furthermore, the growing number and aging of adult CHD patients lead to a spectacular increase in late complications and socioeconomic burden (Dimopoulos et al., 2014; Ford et al., 2015; Marelli et al., 2016; McLeod and Warnes, 2016; Stout et al., 2016). Despite important clinical significance, the etiology responsible for CHD in an overwhelming majority of patients remains unknown. It has been substantiated that CHD is a complex disease and is associated with both environmental and genetic causes (Andersen et al., 2014; Fahed et al., 2013). Well-recognized non-genetic etiologies of CHD include maternal exposures to toxicants, drugs or ionizing radiation during the first trimester of pregnancy and maternal conditions such as viral infection, obesity, diabetes and hypercholesterolemia (Fahed et al., 2013). However, aggregating evidence underscores the genetic origin of familial CHD, which is predominantly transmitted in an autosomal dominant pattern in the family, although familial transmission of CHD also occurs in other inheritance modes, including autosomal recessive and X-linked patterns (Fahed et al., 2013). In addition to chromosomal abnormalities such as chromosome 22q11 deletion and trisomy of chromosome 21, genetic mutations in over 60 genes, including those encoding cardiac transcription factors, cardiac sarcomeric proteins, signaling pathway components and chromatin modifiers, have been implicated in human CHD (Abou Hassan et al., 2015; Andersen et al., 2014; Cao et al., 2016; Chen et al., 2016; Ellesøe et al., 2016; Fahed et al., 2013; Guimier et al., 2015; Homsy et al., 2015; Kassab et al., 2015; Li et al., 2015, 2016; Liu et al., 2016; Lu et al., 2016; Monroy-Muñoz et al., 2015; Pan et al., 2015a,b; Perrot et al., 2015; Postma et al., 2016; Priest et al., 2016; Quintero-Rivera et al., 2015; Sun et al., 2016a,b; Theis et al., 2015a,b; Tong, 2016; Wang et al., 2015; Werner et al., 2016; Yang et al., 2015; Yoshida et al., 2016; Zhang et al., 2016; Zhao et al., 2016; Zheng et al., 2015; Zhou et al., 2016). Among these CHD-causing genes, most code for cardiac transcription factors, including the homeodomain protein NKX2-5, GATA family zinc finger proteins GATA4, GATA 5, and GATA 6, and T-box transcription factors TBX1, TBX5, and TBX20 (Andersen et al., 2014; Fahed et al., 2013; McCulley and Black, 2012). Nevertheless, CHD is a genetically heterogeneous disease, and the genetic defects underlying CHD remain largely elusive. Previous researches have demonstrated the crucial role of cardiac transcription factors in embryonic cardiogenesis and CHD, including the most extensively studied zinc finger transcription factor GATA4 and homeodomain transcription factor NKX2–5 (McCulley and Black, 2012). As another zinc finger transcription factor, CASZ1 is highly expressed in the heart during embryogenesis and essential for proper heart development (Dorr et al., 2015; Liu et al., 2014). In Xenopus embryos, targeted deletion of Casz1 results in abnormal cardiac morphogenesis and embryonic lethality. In mice, disruption of Casz1 leads to aberrant cardiac development, including hypoplasia of myocardium, VSD and disorganized morphology of the heart, which ultimately causes heart failure and embryonic death (Dorr et al., 2015; Liu et al., 2014). In humans, CASZ1 is mapped on chromosome 1p36, and deletion of 1p36, which is the most common telomeric deletion occurring in approximately 1 of 5000 newborns, has been causally linked to congenital cardiovascular deformities and cardiomyopathy, the most common phenotypes of 1p36 deletion syndrome (Zaveri et al., 2014). These findings make it reasonable to scan CASZ1 as a preferred candidate gene for CHD in patients.

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2. Materials and methods 2.1. Study population In this study, a cohort of 172 unrelated patients affected with nonsyndromic CHD was recruited from the Chinese Han population. Among them, there were 97 male cases and 75 female cases with a mean age of 3.4 ± 1.6 years, ranging from 0 to 14 years of age. The available family members of the index patient harboring an identified CASZ1 mutation were also recruited. A total of 200 healthy volunteers, who were excluded from CHD by the cardiac color Doppler echocardiography and were from the same geographic area and ethnicity of the patients, were enrolled as normal controls, and among them, there were 115 male cases and 85 female cases at an average age of 3.3 ± 1.5 years, ranging from 1 to 12 years of age. Diagnosis of CHD was confirmed by cardiac color Doppler echocardiography or cardiac intervention and surgery. Patients with syndromic CHD at the time of recruitment, such as Turner syndrome, Holt-Oram syndrome, Down syndrome, Axenfeld-Rieger syndrome and Di George syndrome, were excluded from the current study. This study was carried out in conformity with the ethical principles outlined in the Declaration of Helsinki. The study protocol was reviewed and approved by the local institutional ethical committee. Informed written consent was obtained from the guardians of the CHD patients and the control individuals prior to commencement of the study. 2.2. Genetic scan of CASZ1 Peripheral venous blood specimens were drawn from all the study participants and stored at −80 °C in polypropylene tubes supplemented with an anticoagulant of ethylene diamine tetraacetic acid. Genomic DNA was isolated from whole blood leukocytes with the QIAamp DNA Mini Kit (Qiagen GmbH, Hilden, Germany) according to the manufacturer's protocol. The concentration and purity of the extracted genomic DNA were measured by an ultraviolet spectrophotometer (Beckman Coulter Inc., Fullerton, CA, USA). The referential genomic DNA sequence of the CASZ1 gene (accession no. NC_000001) was derived from the Nucleotide database at the National Center for Biotechnology Information (http://www.ncbi.nlm.nih.gov/Nucleotide/). With the help of the online program Primer-BLAT (http://www.ncbi.nlm. nih.gov/tools/primer-blast/), the primers to amplify the coding regions and splicing junction sites of CASZ1 by polymerase chain reaction (PCR) were designed as shown in Table 1. Genomic DNA samples

Table 1 Primers for amplification of the coding regions and splicing junction sites of the CASZ1 gene. Coding exon Forward primer (5′ → 3′)

Backward primer (5′ → 3′) Amplicon( bp)

1 2 3-A 3-B 4 5 6 7 8-A 8-B 9, 10 11, 12 13 14 15 16, 17 18-A 18-B

5′-aacagcctcagaagtcgccg-3′ 5′-gtgaggagtcgggtgtgctt-3′ 5′-aagtcgtacttggaggcccg-3′ 5′-aggtgtgtcctcaagcacgg-3′ 5′-ggaggaggcagctaccttgg-3′ 5′-gtcactggcccccaatcgta-3′ 5′-ggctcctgatcaactggggt-3′ 5′-tggatgggacattgggagcc-3′ 5′-ctgttgggaggtttggtggc-3′ 5′-ggatgggggcgaagatcagg-3′ 5′-aatttctctgggtgggggaacg-3′ 5′-aaggaggatgggaagcccac-3′ 5′-gaggaacagggcctcctagc-3′ 5′-gctgcccagcattgagtcac-3′ 5′-agcttgacagtgcctagccc-3′ 5′-ccttctgtcccaagctgcac-3′ 5′-cttgtgcgagtccatctgcg-3′ 5′-gactcggggtccggaagca-3′

5′-gccgagagttgctgccttagt-3′ 5′-gttcgggagggtg ggttgtt-3′ 5′-tagatcgtgtcctgctccgc-3′ 5′-ccagcaccaagaccgaggag-3′ 5′-ggatgtcctcagtcccaccg-3′ 5′-tacagggcccgaagagtcct-3′ 5′-tggacaatctgggctgcctt-3′ 5′-atcctcagcacagacggagc-3′ 5′-cagggccatg gtgtagcagg-3′ 5′-gagtccagcaacgacgacct-3′ 5′-ggagaagcgctgaccagtag-3′ 5′-aggctgccgtgtctgtgttc-3′ 5′-aagttcggaaccaggggacg-3′ 5′-tcttggtggtaccgctggtc-3′ 5′-gagctcttgaaagtcacccgc-3′ 5′-ttactgaccacactgccccc-3′ 5′-aggggtcccatgtgggtgta-3′ 5′-ctcgcacttccactgcacct-3′

453 652 565 669 337 403 434 616 539 685 561 593 625 561 386 641 695 697

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were amplified by PCR using HotStar Taq DNA Polymerase (Qiagen GmbH) on a Veriti Thermal Cycler (Applied Biosystems; Thermo Fisher Scientific Inc., Waltham, MA, USA) under recommended reagent concentrations and standard reaction conditions (Lu et al., 2016). Amplicons were fractionated by electrophoresis on a 1.5% agarose gel and extracted with the QIAquick Gel Extraction Kit (Qiagen GmbH). PCR-sequencing of a purified amplicon was performed with the BigDye® Terminator v3.1Cycle Sequencing Kit (Applied Biosystems; Thermo Fisher Scientific Inc.) on an ABI PRISM 3130 XL DNA Analyzer (Applied Biosystems; Thermo Fisher Scientific Inc.). For an identified CASZ1 sequence variation, a second independent PCR-sequencing analysis was conducted to confirm it. The position of an exonic sequence variation was delineated according to the reference sequence of the CASZ1 mRNA at the Nucleotide database (accession no. NM_001079843.2). Additionally, some public databases for human sequence variations, such as the single nucleotide polymorphism (http://www.ncbi.nlm.nih.gov/SNP), human gene mutation (http:// www.hgmd.org), 1000 Genomes (http://www.1000genomes.org/) and Exome Variant Server (http://evs.gs.washington.edu/EVS) databases, were queried to verify the novelty of an identified CASZ1 sequence variance.

reverse primer: 5′-TGCGCGGCCGCGGGACTCCCGGCTCATC-3′). The amplified fragments were doubly cut by restriction enzymes NheI and NotI (TaKaRa Co.). The digested product with a length of 3588 base pairs was separated by 1.5% agarose gel electrophoresis, purified with the QIAquick Gel Extraction Kit (Qiagen GmbH), and then inserted into the NheI-NotI sites of the pcDNA3.1 vector (Invitrogen; Thermo Fisher Scientific Inc.) to construct a recombinant expression plasmid CASZ1-pcDNA3.1. The identified mutation was introduced into the wild-type CASZ1-pcDNA3.1 plasmid by site-directed mutagenesis with the QuickChange II XL Site-Directed Mutagenesis Kit (Stratagene; Agilent Technologies Inc.) and confirmed by sequencing. Considering that tyrosine hydroxylase (TH) is a known CASZ1 target molecule, and that CASZ1 has recently been validated to activate transcription from the TH promoter in cultured cells (Virden et al., 2012), consequently, we studied whether the CASZ1 variant affected activation of the downstream gene TH in functional analysis. The human TH (accession no. NG_008128) promoter-driven firefly luciferase reporter construct (TH-luc), which contains the proximal 1985-bp 5′-untranslated region of the TH gene, was constructed as described previously (Warner et al., 2009). 2.6. Cell culture, transfection and reporter gene assay

2.3. Alignment of multiple CASZ1 proteins from various species The CASZ1 protein of human was aligned with those of chimpanzee, monkey, dog, cattle, mouse, rat, fowl, zebrafish and frog using the online MUSCLE program (http://www.ebi.ac.uk/Tools/msa/muscle/). 2.4. Prediction of the disease-causing potential of a CASZ1 sequence variation The causative potential of a CASZ1 sequence variation was evaluated by MutationTaster (http://www.mutationtaster.org), PolyPhen-2 (Polymorphism Phenotyping version 2; http://genetics.bwh.harvard.edu/ pph2), PROVEAN (Protein Variation Effect Analyzer; http://provean.jcvi. org/index.php), and SIFT (Sorting Intolerant Form Tolerant; http://sift. jcvi.org/www/SIFT_enst_submit.html). MutationTaster employs a Bayes classifier to eventually predict the pathogenicity of an alteration and calculates probabilities for the alteration to be either a disease-causing mutation or a benign polymorphism. Here the probability value is the probability of the prediction, i.e. a value close to 1 indicates a high ‘security’ of the prediction. PolyPhen-2 is a tool which predicts possible impact of an amino acid substitution on the structure and function of a human protein using straightforward physical and comparative considerations, and the prediction result of PolyPhen-2 is one of the following: benign, possibly damaging and probably damaging. For separation of the deleterious and neutral protein variants (single or multiple amino acid substitutions, insertions, and deletions), PROVEAN introduces a delta alignment score based on the sequence homologs collected from the NCBI NR protein database through BLAST. If the PROVEAN score is equal to or below a predefined threshold (currently set at −2.5), the protein variant is predicted to have a “deleterious” effect; if the PROVEAN score is above the threshold, the variant is predicted to have a “neutral” effect. While the SIFT is based on evolutionary conservation and the outcome ranges from 0 to 1. The amino acid substitution is predicted to be damaging if the score is ≤0.05, and tolerated if the score is N0.05.

Human embryonic kidney (HEK) 293 T cells were grown in Dulbecco's modified Eagle's media supplemented with 10% fetal calf serum as well as 100 μg/ml streptomycin, 100 U/ml penicillin and L-glutamine at 37 °C with 5% CO2. For transient transfection experiments, HEK293T cells were transfected with plasmids using the Lipofectamine 2000® reagent (Invitrogen; Thermo Fisher Scientific Inc.) following the manufacturer's manual. As an internal control, the cytomegalovirus-driven β-galactosidase reporter vector (Promega Corp., Madison, WI, USA) was cotransfected with the TH-luc and CASZ1 expression plasmids to normalize transfection efficiency. The total amount of transfected DNA was always adjusted to 3.0 μg with empty vector pcDNA3.1. Luciferase activity was measured by using the Luciferase Reporter Assay System (Promega Corp.) 36 h after transfection, and β-galactosidase activity was measured concomitantly with the Luminescent Beta Galactosidase Detection Kit II (Clontech Inc., Mountain View, CA, USA) to normalize the luciferase activity. The activity of the TH promoter was expressed as fold activation of firefly luciferase relative to β-galactosidase. Three independent luciferase experiments were performed in triplicates and the results were given as means of three independent experiments. 2.7. Statistical analysis The statistical analyses were made by using the SPSS software package for Windows, version 16.0 (SPSS Inc., Chicago, IL, USA). Continuous variables were expressed as means ± standard deviations (SD). Categorical variables were presented as number and percentage. Comparison of continuous variables between two groups was made using Student's unpaired t-test; while categorical variables were compared with Pearson's χ2 test or Fisher's exact test, as appropriate. A twosided p value b 0.05 indicated statistical difference. 3. Results 3.1. Baseline clinical characteristics of the study participants

2.5. Plasmids and site-directed mutagenesis Human heart cDNAs were prepared by reverse transcription using the SuperScript III first-strand synthesis system (Invitrogen; Thermo Fisher Scientific Inc.), Oligo(dT)20 primer (TaKaRa Co., Dalian, Liaoning, China ) and the total mRNAs (Wei et al., 2013). The wild-type open read frame of the human CASZ1 gene (isoform b; accession no. NM_017766.4) were amplified by PCR using the pfuUltra high-fidelity DNA polymerase (Stratagene; Agilent Technologies Inc., Santa Clara, CA, USA) and a pair of primers (forward primer: 5′-TGCGCTAGCTACATCCGAGGAGGTTC-3′;

In this investigation, a cohort of 172 unrelated patients suffered from CHD was clinically investigated in contrast to a total of 200 unrelated healthy individuals. The patients and controls were matched with respect to geographical region, ethnicity, gender and age. All the patients had echocardiogram-documented CHD, of whom about 9% had a positive family history of CHD. Gender ratio was as expected with more males being affected. The control individuals were apparently healthy with no familial history of CHD or symptoms of cardiovascular diseases, and their echocardiograms showed normal cardiac parameters with no

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Table 2 Clinical characteristics of the patients with congenital heart disease (n = 172). Variable Gender Male Female Age (years) Positive family history of CHD Distribution of various CHDs Isolated CHD VSD ASD PDA DORV TGA CoA TA AS APVD IAA PS ECD Complex CHD TOF VSD + ASD VSD + PDA ASD + PDA DORV + VSD TGA + VSD TA + VSD TOF + ASD Incidence of arrhythmias Atrioventricular block Atrial fibrillation Treatment Surgical repair Percutaneous closure Follow-up

n or mean

% or range

97 75 3 16

56 44 0–14 9

98 37 21 8 6 5 5 4 4 3 2 2 1 74 26 14 10 8 7 5 2 2

57 22 12 5 3 3 3 2 2 2 1 1 1 43 15 8 6 5 4 3 1 1

8 2

5 1

95 46 31

55 27 18

CHD, congenital heart disease; VSD, ventricular septal defect; ASD, atrial septal defect; PDA, patent ductus arteriosus; DORV, double outlet right ventricle; TGA, transposition of the great arteries; CoA, coarctation of the aorta; TA, truncus arteriosus; AS, aortic stenosis; APVD, abnormal pulmonary venous drainage; IAA, interrupted aortic arch; PS, pulmonary stenosis; ECD, endocardial cushion defect; TOF, tetralogy of Fallot.

evidence of structural heart diseases. The baseline clinical features of the studied CHD patients are summarized in Table 2.

Fig. 2. A schematic diagram showing the structural domains of the CASZ1 protein and the location of the mutation associated with congenital heart disease. The mutation identified in patients with congenital heart disease is marked above the structural domains. AA, amino acid; NH2 means amino terminus; NLS, nuclear location signal; ZF, zinc finger; COOH, carboxyl terminus.

position 38 into proline (p. L38P), was detected in a two-year-old girl with VSD and double outlet right ventricle, who had a positive family history of VSD. The DNA sequencing electropherograms showing the heterozygous CASZ1 mutation of c.113 T N C and its wild-type control sequence are displayed in Fig. 1. A schematic drawing of the CASZ1 proteins delineating the key structural domains and location of the mutation identified in this study is shown in Fig. 2. The missense mutation was neither observed in the 200 control subjects nor found in the single nucleotide polymorphism, human gene mutation, 1000 Genomes or Exome Variant Server database (consulted again in August 18, 2016). Genetic scan of the mutation carrier's close relatives available revealed that the mutation was present in all the affected relatives, but absent in unaffected relatives. Analysis of the index patient's pedigree showed that the mutation co-segregated with VSD transmitted as an autosomal dominant trait in the family with complete penetrance. The affected family members had no extra-cardiac manifestations (such as mental retardation or some of the other manifestations of the 1p36 deletion syndrome. The unaffected family members, especially II-2 and III-3 received an echocardiogram to rule out silent CHD. Considering that all the patients and controls sequenced were form the same geographic area, we thought this mutation was likely to be de novo in this family. The pedigree structure of the proband's family is represented in Fig. 3. The phenotypic characteristics of the affected family members available are shown in Table 3. 3.3. Complete conservation of the mutated amino acid Multiple alignments of the CASZ1 protein sequences across species displayed that the mutated residue at amino acid 38 was completely conserved evolutionarily (Fig. 4).

3.2. Identification of a CASZ1 mutation

3.4. CASZ1 sequence variation predicted to be causative

By sequencing of the CASZ1 gene in 172 unrelated patients with CHD, a missense mutation was identified in one patient. Specifically, a substitution of cytosine for thymine at the second nucleotide of codon 38 (c.113 T N C), predicting the transversion of leucine at amino acid

The identified CASZ1 sequence variation was predicted to be disease-causing with a p value of 1.000 by MutationTaster, probably damaging with a score of 1.000 (sensitivity: 0.00; specificity: 1.00) by PolyPhen-2, deleterious with a PROVEAN score of −2.531 by PROVEAN,

Fig. 1. Sequence electropherograms showing the heterozygous CASZ1 mutation and its wild-type control. The arrow indicates the heterozygous nucleotides of T/C in the proband (mutant type) or the homozygous nucleotides of T/T in a control individual (wild type). The rectangle marks the nucleotides comprising a codon of CASZ1.

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Fig. 3. Pedigree structure of the family with congenital heart disease. The family was designated as family 1. Family members are identified by generations and numbers. Square indicates male family member; circle, female member; closed symbol, affected member; open symbol, unaffected member; arrow, proband; “+”, carrier of the heterozygous missense mutation; “−”, non-carrier.

Table 3 Phenotypic characteristics and status of CASZ1 mutation of the affected family members. Individual Family 1 I-1 II-3 II-6 III-2

Gender M M F F

Age (years) 59 31 28 2

Cardiac phenotype

CASZ1 mutation

VSD VSD VSD VSD, DORV

L38P +/− +/− +/− +/−

M, male; F, female; VSD, ventricular septal defect; DORV, double outlet right ventricle; ±, heterozygoty.

and damaging with a SIFT score of 0 and a median information content of 3.84 by SIFT.

3.5. Functional impairment of CASZ1 resulted from the mutation As shown in Fig. 5, the same amount (1.0 μg) of wild-type and L38Pmutant CASZ1-pcDNA3.1 constructs transcriptionally activated the TH promoter by ~11 folds and ~3 fold, respectively. When 0.5 μg of wildtype CASZ1-pcDNA3.1 was used alone or together with 0.5 μg of L38Pmutant CASZ1-pcDNA3.1, the induced activation of the TH promoter was ~ 6 folds or ~ 7 folds. These results indicate that the L38P-mutant CASZ1 has significantly reduced transcriptional activity.

Fig. 5. Functional impairment of CASZ1 caused by the mutation. Activation of the TH promoter driven luciferase in HEK293T cells by wild-type or L38P-mutant CASZ1 (L38P), alone or together, showed significantly diminished transcriptional activation by the mutant protein. Experiments were carried out in triplicate, and means with standard deviations are shown. ** indicates t = 8.56924, p = 0.00102; * indicates t = 4.36841, p = 0.01198, when compared with wild-type CASZ1 (1.0 μg).

4. Discussion In this study, by sequence analysis of CASZ1 in a cohort of CHD patients as well as a mutation carrier's available family members, a novel heterozygous mutation of c.113 T N C, equivalent to p.L38P, was identified in a family with CHD. The missense mutation, which was not detected in the 400 referential chromosomes from a matched control population, co-segregated with CHD in the family with complete penetrance. Biological analyses unveiled that the L38P-mutant CASZ1 protein had significantly decreased transcriptional activity. Hence, it is very likely that mutated CASZ1 contributes to CHD in this family. Nevertheless, we can't exclude the possibility that other genetic variants (common SNPs or rare mutants) may also contribute to CHD in this

Fig. 4. Multiple alignments of CASZ1 proteins among various species. Alignment of multiple CASZ1 proteins across species from human to chimpanzee, monkey, dog, cattle, mouse, rat, fowl, zebrafish and frog exhibited that the altered leucine at amino acid 38 was completely conserved evolutionarily.

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family. Genome-wide association study and whole genome sequencing analysis may be helpful in explaining the possibility for this family, especially for the whole study population. Human CASZ1, which codes for a zinc finger transcription factor, was mapped on chromosome 1p36, and to date, two isoforms of the CASZ1 gene, CASZ1a and CASZ1b, have been cloned (Liu et al., 2006). The fulllength isoform a encompasses 1759 amino acids with 11 zinc fingers; while isoform b comprises 1166 amino acids that are identical to the first 1166 amino acids of isoform a but lacks the last 6 zinc fingers (Liu et al., 2015). The structure and function deciphers have demonstrated that the N-terminus and the first 4 zinc fingers of CASZ1 are required for protein-protein interaction and transcriptional activity (Liu et al., 2015; Virden et al., 2012), and that CASZ1a and CASZ1b function equally in transcriptional activation of several target genes expressed during cardiac morphogenesis, including TH (Liu et al., 2015; Virden et al., 2012). Moreover, CASZ1b has been shown to be the more evolutionarily conserved isoform (Liu et al., 2006, 2015; Virden et al., 2012). Hence, in functional analysis of the mutant CASZ1 protein, we focused on the evolutionarily conserved CASZ1b isoform. As a result, reporter gene assays revealed that the L38P–mutant CASZ1 protein had significantly diminished transcriptional activity but had no dominant-negative effect on its wild-type counterpart. These findings indicate that haploinsufficiency caused by CASZ1 mutation is potentially an alternative pathological mechanism underpinning CHD. Association of CASZ1 loss-of-function mutation with enhanced susceptibility to CHD may be partially ascribed to aberrant heart development. In Xenopus and mammals, expression of Casz1 mRNA was detected throughout the developing heart, which was critical for proper cardiovascular development (Amin et al., 2014). In Xenopus, Casz1-deletion resulted in cardia bifida, due to arrested progenitor cell differentiation along ventral midline (Christine and Conlon, 2008). In Casz1null mice, cardiac developmental anomalies occurred, including VSD, hypoplasia of myocardium, and disorganized morphology of the heart (Liu et al., 2014). Furthermore, cardiac-conditional ablation of Casz1 in mice led to a decreased cardiomyocyte number in both heart fields, a prolonged S phase, a reduced DNA synthesis, and even embryonic lethality (Dorr et al., 2015). In humans, CASZ1 was highly expressed in heart, lung, skeletal muscle, pancreas, testis, small intestine, and stomach, but in the heart, the relative level of CASZ1 expression was the most high (Liu et al., 2006). Although up to now no CASZ1 mutations have been causally linked to CHD in humans, heterozygous deletion of chromosome 1p36, where CASZ1 was located, has been implicated with various CHDs, including VSD, atrial septal defect and non-compaction cardiomyopathy (Jordan et al., 2015; Zaveri et al., 2014). Taken collectively, these results along with the findings of the current study indicate that genetically defective CASZ1 predisposes to CHD in humans. Notably, as seen in the Exome Aggregation Consortium database based on 60,706 exomes (http://exac.broadinstitute.org/gene/ ENSG00000130940), in the gene CASZ1 there were as many as 581 missense mutations in addition to frameshift, nonsense, splice and copy number variants, with a missense mutation prevalence of approximately 1% in the general population. Among these missense mutations there were three, namely p. A36 T, p. A36S and p. S39C near the p. L38P mutation identified in the current study, and all the three missense mutations were predicted to be probably damaging by PolyPhen-2 and deleterious by SIFT, indicating that this structural domain of the CASZ1 protein is functionally important. Nevertheless, the clinical and cardiac echocardiographic characteristics of these mutation carriers were still unclear. In conclusion, this study firstly associates CASZ1 loss-of-function mutation with enhanced susceptibility to CHD in humans, which provides novel insight into the molecular mechanisms of CHD and a potential therapeutic target for CASZ1-linked CHD. Conflict of interest statement The authors have no conflict of interest to state.

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Acknowledgments We are thankful to the study subjects for their dedication to the research. This work was financially supported by grants from the National Natural Science Foundation of China (grant nos. 81470372, 81270161, 81400244 and 81271927), the key program for Basic Research of Shanghai, China (grant no. 14JC1405500), and the key project of Shanghai Chest Hospital, China (grant nos. 2014YZDH10102 and 2014YZDH20500).

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