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Identification and functional characterization of KLF5 as a novel disease gene responsible for familial dilated cardiomyopathy Ruo-Min Dia,b,1, Chen-Xi Yanga,b,1, Cui-Mei Zhaoc,1, Fang Yuand, Qi Qiaoa,b, Jia-Ning Gua,b, Xiu-Mei Lia,b, Ying-Jia Xua,b,e,∗, Yi-Qing Yanga,b,e,f,∗∗ a
Department of Cardiology, The Fifth People's Hospital of Shanghai, Fudan University, Shanghai, China Center for Complex Cardiac Arrhythmias of Minhang District, The Fifth People's Hospital of Shanghai, Fudan University, Shanghai, China c Department of Cardiology, Tongji Hospital, Tongji University School of Medicine, Shanghai, China d Department of Cardiology, Shanghai Tongren Hospital, Shanghai Jiao Tong University School of Medicine, Shanghai, China e Cardiovascular Research Laboratory, The Fifth People's Hospital of Shanghai, Fudan University, Shanghai, China f Central Laboratory, The Fifth People's Hospital of Shanghai, Fudan University, Shanghai, China b
A R T I C LE I N FO
A B S T R A C T
Keywords: Dilated cardiomyopathy Genetics Transcription factor KLF5 Reporter gene analysis
As a prevalent primary myocardial disease, dilated cardiomyopathy (DCM) represents the most common cause of heart failure in the young and the most frequent indication for cardiac transplantation. Aggregating evidence highlights the genetic basis of DCM. However, due to substantial genetic heterogeneity, the genetic defects of DCM in most cases remain elusive. In the current investigation, the entire coding exons and splicing junctions of the KLF5 gene, which encodes a key transcription factor required for cardiac structural and functional remodeling, were sequenced in 234 probands affected with DCM, and a heterozygous KLF5 mutation, NM_001730.5: c.1100T > A; p.(Leu367*), was identified in a proband. Genetic analysis of the proband's family members revealed that the identified KLF5 mutation co-segregated with DCM in the family with complete penetrance. The nonsense mutation was neither detected in 506 control individuals nor reported in such population-genetics databases as ExAC, dbSNP and gnomAD. Biological assays with a dual-luciferase reporter assay system demonstrated that the mutant KLF5 protein had no transcriptional activity when compared with its wildtype counterpart. Furthermore, the mutation abrogated the synergistic transactivation between KLF5 and NFKB1, another pivotal transcription factor that has been causally linked to DCM. The whole-exome sequencing analysis of the proband's family members revealed no other causative genes. The findings indicate KLF5 as a new gene contributing to DCM in humans, implying potential implications for the precision medicine of DCM.
1. Introduction Dilated cardiomyopathy (DCM) is defined as a heart muscle disease characterized by left ventricular cavity dilatation and contractile malfunction in the absence of abnormal pressure or volume overload or coronary artery disease sufficient to explain the systolic impairment (Domínguez et al., 2018; Merlo et al., 2018). As the most common form of primary myocardium disorder, DCM occurs with an estimated prevalence ranging from 1/2500 up to 1/250 people globally, with a 3:1 male to female predominance (Merlo et al., 2018). DCM may contribute to thromboembolism, congestive heart failure, life-threatening supraventricular and ventricular arrhythmias, and sudden cardiac demise,
with the mean 5-year survival rate being approximately 50% after diagnosis of DCM (Anastasakis et al., 2018; Nishimura et al., 2019; Stolfo et al., 2018; Tabish et al., 2017; Weintraub et al., 2017). Factually, DCM remains the most common cause of heart failure in the young and the most prevalent indication for heart transplantation in both children and adults worldwide (Tabish et al., 2017; Weintraub et al., 2017). Despite significant clinical importance, the molecular etiologies underlying DCM are largely obscure. Aggregating evidence has convincingly demonstrated the pivotal role of genetic defects in the pathogenesis of DCM, especially for familial DCM (McNally and Mestroni, 2017). Recent studies suggest that over 50% of patients with DCM have a strong genetic predisposition to
∗
Corresponding author. Department of Cardiology, The Fifth People's Hospital of Shanghai, Fudan University, 801 Heqing Road, Shanghai, 200240, China. Corresponding author. Cardiovascular Research Laboratory, The Fifth People's Hospital of Shanghai, Fudan University, 801 Heqing Road, Shanghai, 200240, China. E-mail addresses:
[email protected] (Y.-J. Xu),
[email protected] (Y.-Q. Yang). 1 These authors contributed equally to the work. ∗∗
https://doi.org/10.1016/j.ejmg.2019.103827 Received 16 September 2019; Received in revised form 25 October 2019; Accepted 14 December 2019 1769-7212/ © 2019 Elsevier Masson SAS. All rights reserved. This is an open access article under the#lictext# license (http://creativecommons.org/licenses/#lictextcc#/#licvalue#/).
Please cite this article as: Ruo-Min Di, et al., European Journal of Medical Genetics, https://doi.org/10.1016/j.ejmg.2019.103827
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2018, among whom 78 (33%) patients had a positive family history for DCM. The available family members of the index patients were also recruited. A total of 506 unrelated healthy subjects were enlisted as controls. All the study participants were from the Chinese Han-race population. For each study individual, detailed clinical investigation was performed, including familial and medical histories, physical examination, resting 12-lead electrocardiogram, and transthoracic echocardiography with color Doppler. The patients with DCM also underwent coronary angiography and exercise performance test, but the endo-myocardial biopsy and cardiac magnetic resonance imaging were conducted only if there was a strong indication. DCM was defined according to the criteria established by the World Health Organization/ International Society and Federation of Cardiology Task Force on the Classification of Cardiomyopathy: a left ventricular end-diastolic diameter > 27 mm/m2 and an ejection fraction < 40% or a fractional shortening < 25% in the absence of abnormal loading conditions, coronary artery disease, congenital heart disease, and other systemic diseases (Liu et al., 2019; Xu et al., 2019). Patients were excluded from the current study if they had comorbidities that may cause cardiac contractile dysfunction, such as arterial hypertension, coronary artery disease, and valvular heart disease. The disease was defined as familial when two or more persons in the family met the same diagnostic criteria for DCM as index patient. Clinical studies were carried out with investigators blinded to the results of genetic researches. This study was performed in accordance with the principles of the Declaration of Helsinki. The study protocol was approved by the ethics committee of the Fifth People's Hospital of Shanghai, Fudan University, China. Written informed consent was obtained from all individuals studied prior to investigation.
the disease, and most patients with familial DCM present autosomaldominant inheritance, though autosomal recessive, X-linked, and mitochondrial transmission are also observed (McNally and Mestroni, 2017). So far, > 60 genes have been identified to cause DCM, of which the majority code for sarcomeric, desmosomal, cytoskeletal and nuclear envelope proteins where pathogenic genetic mutations impair the generation and transmission of myocardial systolic force, resulting in cardiac systolic dysfunction and congestive heart failure (Aspit et al., 2019; Begay et al., 2018; Chen et al., 2019; Gourzi et al., 2019; Horvat et al., 2019; Iuso et al., 2018; Kawakami et al., 2018; Lee et al., 2019; Li et al., 2019; McNally and Mestroni, 2017; Minoche et al., 2019; Nozari et al., 2018; Pankuweit, 2018; Shakeel et al., 2018; Streff et al., 2018; van den Hoogenhof et al., 2018). Nevertheless, these well-established DCM-causing genes only account for a small portion of DCM, and the genetic determinants underpinning DCM in the vast majority of cases remain to be discovered. Recently, emerging evidence indicates that cardiac core transcription factors have crucial roles in the embryonic cardiac morphogenesis and postnatal heart adaptation (Oka et al., 2007), and mutations in multiple transcription factors, including GATA4 (Li et al., 2013), GATA6 (Xu et al., 2014), MEF2C (Yuan et al., 2018), HAND1 (Firulli et al., 2019), HAND2 (Liu et al., 2019), and ISL1 (Xu et al., 2019), have been causally linked to DCM. As another cardiac key transcription factor, Krüppel-like factor 5 (KLF5) is highly expressed in the cardiovascular system during embryogenesis and throughout life, where it transcriptionally mediates the expression of several genes essential for proper cardiac structure and function, including those encoding peroxisome proliferator-activated receptor α (PPAR1), platelet‒derived growth factor subunit A (PDGFA), transforming growth factor‒β (TGFβ), vascular endothelial growth factor A (VEGFA), insulin like growth factor 1 (IGF1), and myosin heavy chain (Aizawa et al., 2004; Drosatos et al., 2016; Nagai et al., 2005; Takeda et al., 2010). Notably, KLF5 may physically interact with its transcriptionally-cooperated partners, such as nuclear factor-kappa B subunit 1 (NFKB1), GATA4 and GATA6, to synergistically activate the transcription of target genes (Aizawa et al., 2004; Chia et al., 2015). Previous investigations underscore the critical role of KLF5 in regulating various cellular biological processes, encompassing proliferation, migration, differentiation, growth, survival and apoptosis (Dong and Chen, 2009). In mice, homozygous deletion of Klf5 led to early embryonic lethality; whereas heterozygous Klf5-knockout mice showed diminished levels of angiogenesis, cardiac hypertrophy and interstitial fibrosis in response to angiotensin II infusion (Shindo et al., 2002). Additionally in mice, haploinsufficiency due to cardiac fibroblast-specific deletion of Klf5 ameliorated cardiac hypertrophy and fibrosis elicited by moderate-intensity pressure overload, while high-intensity pressure overload resulted in severe heart failure and early death in mice with Klf5-null fibroblasts (Takeda et al., 2010). Furthermore, cardiomyocyte-specific Klf5-knockout mice showed reduced expression of cardiac PPAR1 and its downstream fatty acid metabolism-related targets, which gave rise to decreased cardiac fatty acid oxidation, ATP levels, increased triglyceride accumulation, and cardiac dysfunction (Drosatos et al., 2016). By contrast, overexpression of KLF5 significantly enhanced cell activity and decreased cell apoptosis in response to myocardial injury (Li et al., 2016). These findings underscore the pivotal role of KLF5 in cardiovascular adaptive remodeling, and make it justifiable to screen KLF5 as a prime candidate gene for human DCM. The current research was sought to identify KLF5 mutations responsible for DCM and reveal underlying mechanism by which mutant KLF5 contributes to DCM.
2.2. Genetic analysis Approximately 2 mL of peripheral blood samples were collected in citrate-coated tubes from the study participants. Genomic DNA was isolated from whole blood using the GeneJET Genomic DNA Purification Kit (Thermo Fisher Scientific, Waltham, MA, USA) following the manual. The entire coding exons and flanking intronic sequences of the KLF5 gene were sequenced in 234 unrelated patients with DCM. The available relatives of the proband harboring an identified KLF5 mutation and 506 unrelated control individuals were genotyped for KLF5. The primers used to amplify the coding regions and splicing junctions of KLF5 by polymerase chain reaction (PCR) were designed as shown in Table S1. Amplification of specific genomic DNA fragments by PCR was performed using HotStar Taq DNA Polymerase (Qiagen, Hilden, Germany) on a Veriti Thermocycler (Applied Biosystems, Foster, CA, USA) with recommended concentrations of reagents. Amplicons were purified with the QIAquick Gel Extraction Kit (Qiagen), and PCR-sequenced with the BigDye Terminator Cycle Sequencing Kit (Applied Biosystems) on the ABI PRISM 3130 XL DNA Analyzer (Applied Biosystems) following the manufacturer's protocol. For a confirmed KLF5 sequence variant, the Exome Aggregation Consortium database (ExAC; http://exac.broadinstitute.org/), the Single Nucleotide Polymorphism database (dbSNP; http://www.ncbi.nlm.nih.gov/SNP) and the Genome Aggregation Database (gnomAD; http://gnomad. broadinstitute.org/) were consulted to verify its novelty. In addition, in Family 1 with an identified KLF5 mutation, wholeexome sequencing analysis of the family members was performed as previously described (Xu et al., 2019) to rule out the potential causative roles of other genes. In brief, 5 μg of genomic DNA from each family member was utilized to construct an exome library with the SureSelect Human All Exon V5 Kit (Agilent Technologies, Santa Clara, CA, USA) and was sequenced under the Genome Analyzer platform (Illumina, San Diego, CA, USA), following the manufacturer's protocol. Raw image files were processed by Illumina Pipeline for base calling with default parameters, and generating the reads set. SOAPaligner was used to map the reads on the human reference genome. SNPs and insertions/
2. Subjects and methods 2.1. Study participants In the present study, 234 unrelated patients with DCM were consecutively registered between February 18, 2015 and November 30, 2
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NFKB1-pcDNA3.1, in combination with 1.0 μg of PDGFA-luc and 0.04 μg of pGL4.75. Transfected cells were incubated for 36 h, then harvested and lysed. Firefly luciferase and Renilla luciferase activities of the cell lysates were measured on the GloMax-96 luminometer (Promega) with the Dual-Glo Luciferase Assay System (Promega) according to the manufacturer's descriptions. The activity of the VEGFA or PDGFA promoter was shown as fold activation of Firefly luciferase relative to Renilla luciferase. For each plasmid transfection, three independent experiments were conducted in triplicates, and the representative results were given as mean and standard deviation of three independent experimental results.
deletions (indels) were reported by SAMtools. The identified sequence variants in known genes were categorized according to the guidelines described previously (Richards et al., 2015). The pathogenic mutations identified by exome sequencing were verified by segregation analysis through di-deoxy sequencing. 2.3. Plasmid constructs and site-targeted mutagenesis Total RNA extraction from human heart specimen and cDNA synthesis by reverse transcription-PCR were described previously (Wang et al., 2019). The full-length wild-type cDNA of the human KLF5 gene (accession no. NM_001730.5) was produced by PCR using the pfuUltra high-fidelity DNA polymerase (Stratagene, Santa Clara, CA, USA) with a specific pair of primers (forward primer: 5′-GTAGAATTC GACCCGCGCCTGGAGCTGCG-3′; reverse primer: 5′-GTAGCGGCCGCA ACGGGTCACACGGGCAGTG-3′). The amplified cDNA of KLF5 was doubly cut by restriction enzymes EcoRI and NotI (NEB, Hitchin, Herts, UK), purified with QIAquick Gel Extraction Kit (Qiagen), and inserted at the EcoRI-NotI sites into the pcDNA3.1 plasmid (Invitrogen, Carlsbad, CA, USA) to construct a eukaryotic expression plasmid KLF5-pcDNA3.1. The Leu367*-mutant KLF5-pcDNA3.1 was generated by PCR-based sitedirected mutagenesis using the Quick Change II XL Site-Directed Mutagenesis Kit (Stratagene, La Jolla, CA, USA) and a complementary pair of primers (forward primer: 5′-GGAGTAACCCCGATTAGGAGAAACGA CGCAT-3′; reverse primer: 5′-ATGCGTCGTTTCTCCTAATCGGGGTTAC TCC-3′), and was validated by direct sequencing after the wild-type KLF5-pcDNA3.1 template was digested by DpnI (NEB). Similarly, the full-length wild-type cDNA of the human NFKB1 gene (accession no. NM_003998.4) was prepared, and inserted at the EcoRI-NotI sites into the pcDNA3.1 plasmid (Invitrogen) to construct the NFKB1-pcDNA3.1 plasmid. To create the reporter plasmid VEGFA-luciferase (VEGFA-luc), which expresses Firefly luciferase, a 1718-bp promoter region of the VEGFA gene (nucleotides from −1618 to +100, with initial transcription nucleotide numbered +1; accession No. NG_008732.1) was sub-cloned into the pGL3-Basic vector lacking eukaryotic promoter and enhancer (Promega, Madison, WI, USA) as described previously (Gao et al., 2015). For construction of the reporter plasmid PDGFA-luc, which expresses Firefly luciferase, a 640-bp promoter region of the human PDGFA gene (nucleotides from −630 to +10; accession No. NG_029727.1) was sub-cloned into the promoter-free pGL3-Basic plasmid (Promega) as described previously (Aizawa et al., 2004).
2.5. Statistical analysis Data for continuous variables with normal distribution were presented as mean with standard deviation, and for categorical variables were expressed as numbers and percentages. The Pearson's χ2 test, or Fisher's exact test when indicated, was used for comparison of categorical variables between patient and control groups. The unpaired Student's t-test was used to compare numeric variables between two groups. When comparison among multiple groups was performed, oneway analysis of variance, followed by Fisher's protected least significant difference test was used. When difference is significant, subsequent analysis with post hoc t-test with Bonferroni correction was performed. For each test, a two-tailed p < 0.05 indicated significant difference. All statistical analyses were made with the SPSS for Windows software package, version 17.0 (SPSS, Chicago, IL, USA). 3. Results 3.1. Baseline clinical characteristics of the study subjects A consecutive cohort of 234 unrelated DCM patients was clinically investigated in contrast to a total of 506 unrelated healthy subjects. The patients were matched with the healthy controls for age, gender and ethnicity. All the study subjects came from the same geographical area, and had no traditional risk factors for DCM. The patients manifested with congestive heart failure, and treated with angiotensin-converting enzyme inhibitors or angiotensin-II type-1 receptor blockers, aldosterone receptor antagonists and β-adrenergic blockers. Each patient had echocardiogram-documented heart failure, but no secondary causes of heart failure, especially for coronary artery disease, which was excluded by routine coronary angiography. The control individuals had neither evidence of heart disease nor family history of DCM, and their echocardiographic images were normal. The baseline clinical features of the study participants are summarized in Table 1.
2.4. Cell culture, transfection, and reporter gene assay HeLa and COS-7 cells were grown in Dulbecco's modified Eagle's medium (Invitrogen) containing 10% fetal bovine serum (Invitrogen), 100 μg/mL streptomycin (Sigma-Aldrich, St. Louis, MO, USA) and 100U/mL penicillin (Sigma-Aldrich) in an incubator at 37 °C with 5% CO2. Cells were seeded in 6-well plates 24 h before transfection, at a density of 2 × 105 cells in 1 mL cell culture medium per well. Cellular transient transfections with various expression plasmids were carried out using the FuGENE HD transfection reagent (Promega) according to the manufacturer's specifications. The internal control reporter plasmid pGL4.75 (Promega), which expresses Renilla luciferase, was utilized in transfection analysis to normalize transfection efficiency. Specifically, COS-7 cells were transfected with 0.8 μg of empty pcDNA3.1, or wildtype KLF5-pcDNA3.1, or Leu367*-mutant KLF5-pcDNA3.1, or 0.4 μg of empty pcDNA3.1 plus 0.4 μg of wild-type KLF5-pcDNA3.1, or 0.4 μg of wild-type KLF5-pcDNA3.1 plus 0.4 μg of Leu367*-mutant KLF5pcDNA3.1, in the presence of 1.0 μg of VEGFA-luc and 0.04 μg of pGL4.75. To analyze the synergistic transcriptional activation between KLF5 and NFKB1 (Firulli et al., 2019), HeLa cells were transfected with 1.0 μg of empty pcDNA3.1, or 1.0 μg of NFKB1-pcDNA3.1, or 1.0 μg of wild-type KLF5-pcDNA3.1, or 1.0 μg of Leu367*-mutant KLF5pcDNA3.1, or 1.0 μg of wild-type KLF5-pcDNA3.1 plus 1.0 μg of NFKB1pcDNA3.1, or 1.0 μg of Leu367*-mutant KLF5-pcDNA3.1 plus 1.0 μg of
3.2. Identification of KLF5 mutation By sequence analysis of the coding regions and splicing sites of the KLF5 gene, a heterozygous mutation was detected in 1 out of 234 unrelated patients affected with DCM, with an estimated mutational prevalence of 0.43%. Specifically, a substitution of adenine for thymine in the second nucleotide of codon 367, predicting the transition of the codon encoding leucine at amino acid 367 into a premature stop codon, namely NM_001730.5: c.1100T > A; p.(Leu367*), was discovered in the proband from family 1. This KLF5 variant was submitted to the ClinVar database (https://www.ncbi.nlm.nih.gov/clinvar/) with an accession number of SCV000996312. The chromatograms showing the heterozygous KLF5 mutation of c.1100T > A as well as its wild-type control sequence are illustrated in Fig. 1A. A schematic diagram displaying the structural domains of the wild-type and mutant KLF5 proteins is shown in Fig. 1B. The nonsense mutation was neither observed in the control people nor reported in the ExAC, dbSNP and gnomAD databases (queried again on September 16, 2019). Of note, in genetic DCM penetrance is always age-dependent. Considering that the 3
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Table 1 Demographic and clinical characteristics of the study subjects. Parameters
Patients (n = 234)
Age (years) 47 ± 14 Male (%) 136 (58) Positive family history of 78 (33) DCM (%) HR (bpm) 75 ± 13 SBP (mmHg) 117 ± 14 DBP (mmHg) 77 ± 9 LVEDD (mm) 70 ± 8 LVESD (mm) 59 ± 7 LVEF (%) 37 ± 7 LVFS (%) 21 ± 5 NYHA cardiac function class (%) I 30 (13) II 82 (35) III 96 (41) IV 26 (11)
Controls (n = 506)
p-Value
48 ± 15 297 (59) 0 (0)
0.3895 0.8824 < 0.0001
74 ± 11 118 ± 13 78 ± 7 41 ± 6 32 ± 5 63 ± 6 34 ± 4
0.2787 0.3427 0.1003 < 0.0001 < 0.0001 < 0.0001 < 0.0001
NA NA NA NA
NA NA NA NA
DBP: diastolic blood pressure; DCM: dilated cardiomyopathy; HR: heart rate; LVEDD: left ventricular end-diastolic diameter; LVEF: left ventricular ejection fraction; LVESD: left ventricular end-systolic diameter; LVFS: left ventricular fractional shortening; NA: not applicable; NYHA: New York Heart Association; SBP: systolic blood pressure.
youngest affected persons were in fact 28 and 30 years old, respectively, they were indeed rather young even for genetic DCM. It might also be worth mentioning that the probability of being loss-of-function intolerant (pLI) for KLF5 was 0.82, suggesting that there is some evolutionary constraint on loss of function mutations in KLF5 possibly because patients tend to develop cardiomyopathy before they reproduce. By comparison with the fact that pLI for other known autosomal loss-of-function cardiomyopathy genes like MYBPC3, TTN and PKP2 are 0.59, 0 and 0 respectively, indeed we shouldn't expect the majority of carriers of premature stop codons in those genes to become seriously ill before reproductive age. Genetic screening of the family demonstrated that the mutation was present in all living family members affected with DCM, but absent in unaffected family members examined. Analysis of the pedigree showed that the mutation co-segregated with DCM, which was transmitted in an autosomal dominant pattern in the family with complete penetrance. The pedigree structure of the family is exhibited in Fig. 1C. The phenotypic characteristics of the affected family members alive from this family are presented in Table 2. Additionally, genomic DNA samples from three affected family members (II-3, III-1 and III-2) and one unaffected family member (II-4) of the proband carrying an identified KLF5 mutation were subjected to whole-exome sequencing analysis, yielding an average of 11 Gb data with an approximately 96% coverage of target region and a 94% of target covered over 10×. An average of 7832 missense, nonsense, and splice site variants (range 7039–8518) was identified for each subject, of which 12 variants with minor allele frequency < 0.01 (from the ExAC, dbSNP and gnomAD databases) were predicted to be causative by three online programs (MutationTaster, PolyPhen-2, and SIFT), and shared by all three affected subjects in heterozygous status. After filtering with cardiomyopathy-related genes in combination with cosegregation analysis, only the KLF5 gene mutation co-segregated with DCM in the family, highlighting the disease-causing role of mutant KLF5. Although other 11 detected variants were filtered out by absence from the plausible or known DCM-related genes, we couldn't rule out the possibility that such additional genetic abnormalities, which had a much smaller effect, might very well play a role in the 100% penetrance in relatively young patients (probably in a di-genic way).
Fig. 1. KLF5 mutation linked to dilated cardiomyopathy. (A) DNA sequence electropherograms illustrating the KLF5 mutation as well as its wild-type control. The arrow points to the heterozygous nucleotides of T/A in the proband (mutant) or the homozygous nucleotides of T/T in a healthy control individual (wild-type). The rectangle marks the nucleotides comprising a codon of KLF5. (B) A schematic diagram displaying the structural domains of the wild-type and Leu367*-mutant KLF5 proteins. The mutation linked to dilated cardiomyopathy was noted above the domain. NH2 denotes amino-terminus; TAD, transcriptional activation domain; ZF, zinc finger; COOH, carboxyl-terminus. (C) Pedigree structure of the family affected with dilated cardiomyopathy. The family with KLF5 mutation linked to dilated cardiomyopathy was arbitrarily designated as Family 1. Family members are recognized by generations and numbers.
3.3. Diminished transcriptional activity of the mutant KLF5 As shown in Fig. 2, the same amount (0.8 μg) of wild-type and Leu367*-mutant KLF5-pcDNA3.1 transcriptionally activated the VEGFA promoter by ~15 folds and ~1 fold, respectively. When the same amount (0.4 μg) of wild-type KLF5-pcDNA3.1 was co-transfected with empty pcDNA3.1 or Leu367*-mutant KLF5-pcDNA3.1, the induced transactivation of the VEGFA promoter was ~9-fold or ~7-fold. These data suggest that Leu367*-mutant KLF5 has no transcriptional activity or predominant negative effect on its wild-type counterpart. 3.4. Abrogated synergistic transcriptional activation between NFKB1 and mutant KLF5 As shown in Fig. 3, the same amount (1.0 μg) of wild-type and Leu376*-mutant KLF5-pcDNA3.1 activated the PDGFA promoter by ~8 folds and ~1 fold, respectively. In the presence of 1.0 μg of wild-type 4
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Table 2 Phenotypic features and KLF5-mutation status of the living family members with dilated cardiomyopathy. Individual
Gender
Age (years)
Cardiac phenotype
LVEDD (mm)
LVESD (mm)
LVEF (%)
LVFS (%)
KLF5 mutation (Leu367*)
II-3 II-8 II-9 III-1 III-2
M F M M F
51 46 42 30 28
DCM, LBBB DCM, PVB DCM DCM DCM
75 61 66 66 58
65 53 56 54 44
27 31 39 41 48
13 15 16 19 23
+/− +/− +/− +/− +/−
DCM: dilated cardiomyopathy; F: female; LBBB: left bundle branch block; LVEDD: left ventricular end-diastolic diameter; LVEF: left ventricular ejection fraction; LVESD: left ventricular end-systolic diameter; LVFS: left ventricular fractional shortening; M: male; PVB: premature ventricular beat; +/−: heterozygote.
NFKB1-pcDNA3.1, the same amount (1.0 μg) of wild-type and Leu376*mutant KLF5-pcDNA3.1 activated the PDGFA promoter by ~28 folds and ~7 folds, respectively, indicating that the Leu367*-mutant KLF5 protein loses transcriptional activity and the mutation disrupts the synergistic transactivation between KLF5 and NFKB1. 4. Discussion In the present research, a heterozygous KLF5 mutation, NM_001730.5: c.1100T > A; p.(Leu367*), was identified in a family suffering from DCM. The nonsense mutation co-segregated with DCM in the family with complete penetrance. The mutation was neither found in the 1012 reference chromosomes from the control individuals nor reported in the ExAC, dbSNP and gnomAD databases. Functional analyses demonstrated that the Leu376*-mutant KLF5 protein had no transcriptional activity. Furthermore, the mutation disrupted the synergistic transactivation between KLF5 and NFKB1. Additionally, whole-exome sequencing analysis showed that only the KLF5 mutation co-segregated with DCM in the family. Hence, it is very probable that genetically compromised KLF5 predisposes to DCM in these mutation carriers. In humans, KLF5, also termed BTEB2, CKLF or IKLF, maps on chromosome 13q22.1, coding for a transcription factor protein with 457 amino acids. Previous investigations have substantiated that KLF5, alone or in synergy with its transcriptionally cooperated partners, such as GATA4, GATA6 and NFKB1, regulates expression of multiple target genes in the heart, including TGFβ, PPAR1 and IGF1, which play critical roles in cardiac structural remodeling and functional adaptation (Aizawa et al., 2004; Drosatos et al., 2016; Nagai et al., 2005; Takeda et al., 2010). Specifically, in animal models of cardiac hypertrophy, and in patients with hypertrophic cardiomyopathy, myocardial expression of TGFβ was significantly upregulated (Dobaczewski et al., 2011). Furthermore, in the murine heart, overexpression of TGFβ led to cardiac hypertrophy and fibrosis (Dobaczewski et al., 2011). In addition, TGFβ signaling is also essential for maintaining cardiac function, sarcomere kinetics, ion-channel gene expression, and cardiomyocyte survival (Umbarkar et al., 2019). By contrast, inhibition of TGFβ signaling contributed to DCM (Lucas et al., 2010; Umbarkar et al., 2019). PPAR1 was required for proper cardiac energy metabolism, and decreased PPRA1 was associated with diminished cardiac fatty acid oxidation, ATP levels, increased triglyceride accumulation, and cardiac dysfunction (Drosatos et al., 2016). In a transgenic mouse model of DCM, cardiac-specific expression of IGF1 substantially extended the lifespan of the transgenic mice, markedly improved cardiac functions, and delayed DCM and heart failure (Touvron et al., 2012). By contrast, mice with combined deficiency of the insulin receptor and IGF1 receptor in the heart developed early-onset DCM and died from heart failure within the first month of life (Laustsen et al., 2007). Importantly, pathogenic mutations in GATA4 and GATA6 have been identified to be responsible for DCM in humans (Li et al., 2013). In addition, there is also evidence linking reduced NFKB1 expression to heart failure and DCM (Zhou et al., 2009). Taken collectively, these observational results together with those mentioned above (Dong and Chen, 2009; Drosatos et al., 2016; Li et al., 2016; Shindo et al., 2002; Takeda et al., 2010) indicate
Fig. 2. Functional failure of Leu367*-mutant KLF5. In cultured COS-7 cells, in contrast to wild-type KLF5, Leu367*-mutant KLF5 had no transactivation of luciferase reporter driven by the promoter of vascular endothelial growth factor A. Experiments were done in triplicates, with mean and standard deviations shown. ## and # represent t = 9.83304, p = 0.00060 and t = 4.85153, p = 0.00833, respectively, when compared with wild-type KLF5.
Fig. 3. Disrupted synergistic transactivation between KLF5 and NFKB1 by the mutation. In cultured HeLa cells, the synergistic activation of the promoter of platelet‒derived growth factor subunit A by NFKB1 and Leu367*-mutant KLF5 was significantly decreased when compared with that by NFKB1 and wild-type KLF5. Experiments were conducted in triplicates, with mean and standard deviations shown. ## represents t = 9.65431, p = 0.00064 and # represents t = 12.5649, p = 0.00023, when compared with their wild-type counterparts. One-way analysis of variance, followed by the Fisher's protected least significant difference test, was used for multiple comparisons (among KLF5, Leu367*, NFKB1, KLF5 + NFKB1 and Leu367* + NFKB1), and significant statistical difference was indicated (F = 171.189, p = 3.66×10−9). Comparisons were made between KLF5 and Leu367* (t = 7.2467, p = 0.00084), between KLF5 and KLF5 + NFKB1 (t = 19.9033, p < 0.00001), between Leu367* and Leu367* + NFKB1 (t = 6.0167, p = 0.00344), between NFKB1 and KLF5 + NFKB1 (t = 26.0367, p < 0.00001), between NFKB1 and Leu367* + NFKB1 (t = 4.9033, p = 0.01367), and between KLF5 + NFKB1 and Leu367* + NFKB1 (t = 21.1333, p < 0.00001).
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that KLF5 haploinsufficiency is an alternative molecular mechanism underpinning DCM. Previous experiments have validated that a premature termination codon may elicit degradation of mRNA in a great variety of organisms and cell lines by an mRNA quality-control mechanism termed nonsensemediated mRNA decay, thereby preventing the production of truncated protein that may cause disease in humans (Kurosaki and Maquat, 2016). Given the truncating character of the mutation we found, the disease causing mechanism is most likely to be KLF5 haploinsufficiency, although we cannot exclude an additional dominant negative effect of small amounts of translated truncated KLF5 protein (Kurosaki and Maquat, 2016; Ma et al., 2019; Qiao et al., 2018). In conclusion, the current research firstly indicates KLF5 as a new gene predisposing to DCM, which provides novel insight into the molecular pathogenesis of DCM, suggesting potential implications for early prophylaxis and personalized management of DCM in humans.
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Declaration of competing interest The authors declare no conflict of interests. Acknowledgments The authors are thankful to the study participants for their participation in the study. This work was financially supported in part by the grants from the National Natural Science Foundation of China (No. 81470372 to Y.-Q. Yang and No. 81600228 to C.-M. Zhao), the Medicine Guided Program of Shanghai, China (No. 19411971900 to Y.J. Xu), the Clinical Medicine Program of Shanghai, China (19401970200 to Y.-J.X.), the Program of Health and Family Planning Commission of Shanghai, China (No. 20154Y0026 to C.-M. Zhao), the Natural Science Foundation of Minhang District, Shanghai, China (No. 2018MHZ072 to Q. Qiao and No. 2019MHZ014 to C.-X. Yang), the Acute Heart Failure Specialty Program of Health and Family Planning Commission of Changning District, Shanghai, China (No. 20162002 to F. Yuan), and the Key Project of the Fifth People’s Hospital of Shanghai, Fudan University, China (No. 2018WYZD05 to Y.-J. Xu). Appendix A. Supplementary data Supplementary data to this article can be found online at https:// doi.org/10.1016/j.ejmg.2019.103827. References Aizawa, K., Suzuki, T., Kada, N., Ishihara, A., Kawai-Kowase, K., Matsumura, T., et al., 2004. Regulation of platelet-derived growth factor-A chain by Krüppel-like factor 5: new pathway of cooperative activation with nuclear factor-kappa B. J. Biol. Chem. 279, 70–76. Anastasakis, A., Papatheodorou, E., Ritsatos, K., Protonotarios, N., Rentoumi, V., Gatzoulis, K., et al., 2018. Sudden unexplained death in the young: epidemiology, aetiology and value of the clinically guided genetic screening. Europace 20, 472–480. Aspit, L., Levitas, A., Etzion, S., Krymko, H., Slanovic, L., Zarivach, R., et al., 2019. CAP2 mutation leads to impaired actin dynamics and associates with supraventricular tachycardia and dilated cardiomyopathy. J. Med. Genet. 56, 228–235. Begay, R.L., Graw, S.L., Sinagra, G., Asimaki, A., Rowland, T.J., Slavov, D.B., et al., 2018. Filamin C truncation mutations are associated with arrhythmogenic dilated cardiomyopathy and changes in the cell-cell adhesion structures. JACC Clin. Electrophysiol. 4, 504–514. Chen, S.N., Lombardi, R., Karmouch, J., Tsai, J.Y., Czernuszewicz, G., Taylor, M.R.G., et al., 2019. DNA damage response/TP53 pathway is activated and contributes to the pathogenesis of dilated cardiomyopathy associated with LMNA (lamin A/C) mutations. Circ. Res. 124, 856–873. Chia, N.Y., Deng, N., Das, K., Huang, D., Hu, L., Zhu, Y., et al., 2015. Regulatory crosstalk between lineage-survival oncogenes KLF5, GATA4 and GATA6 cooperatively promotes gastric cancer development. Gut 64, 707–719. Dobaczewski, M., Chen, W., Frangogiannis, N.G., 2011. Transforming growth factor (TGF)-β signaling in cardiac remodeling. J. Mol. Cell. Cardiol. 51, 600–606. Domínguez, F., Cuenca, S., Bilińska, Z., Toro, R., Villard, E., Barriales-Villa, R., et al., 2018. Dilated cardiomyopathy due to BLC2-associated athanogene3 (BAG3) mutations. J. Am. Coll. Cardiol. 72, 2471–2481. Dong, J.T., Chen, C., 2009. Essential role of KLF5 transcription factor in cell proliferation
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