Empagliflozin ameliorates vascular calcification in diabetic mice through inhibiting Bhlhe40-dependent NLRP3 inflammasome activation.

Type 2 diabetes mellitus (T2DM) patients exhibit greater susceptibility to vascular calcification (VC), which has a higher risk of death and disability. However, there is no specific drug for VC therapy. NLRP3 inflammasome activation as a hallmark event of medial calcification leads to arterial stiffness, causing vasoconstrictive dysfunction in T2DM. Empagliflozin (EMPA), a sodium-glucose co-transporter 2 inhibitor (SGLT2i), restrains hyperglycemia with definite cardiovascular benefits. Given the anti-inflammatory activity of EMPA, herein we investigated whether EMPA protected against VC in the aorta of T2DM mice by inhibiting NLRP3 inflammasome activation. Since db/db mice receiving a normal diet developed VC at the age of about 20 weeks, we administered EMPA (5, 10, 20 mg·kg-1·d-1, i.g) to 8 week-old db/db mice for 12 weeks. We showed that EMPA intervention dose-dependently ameliorated the calcium deposition, accompanied by reduced expression of RUNX2 and BMP2 proteins in the aortas. We found that EMPA (10 mg·kg-1·d-1 for 6 weeks) also protected against VC in vitamin D3-overloaded mice, suggesting the protective effects independent of metabolism. We showed that EMPA (10 mg·kg-1·d-1) inhibited the abnormal activation of NLRP3 inflammasome in aortic smooth muscle layer of db/db mice. Knockout (KO) of NLRP3 significantly alleviated VC in STZ-induced diabetic mice. The protective effects of EMPA were verified in high glucose (HG)-treated mouse aortic smooth muscle cells (MOVASs). In HG-treated NLRP3 KO MOVASs, EMPA (1 µM) did not cause further improvement. Bioinformatics and Western blot analysis revealed that EMPA significantly increased the expression levels of basic helix-loop-helix family transcription factor e40 (Bhlhe40) in HG-treated MOVASs, which served as a negative transcription factor directly binding to the promotor of Nlrp3. We conclude that EMPA ameliorates VC by inhibiting Bhlhe40-dpendent NLRP3 inflammasome activation. These results might provide potential significance for EMPA in VC therapy of T2DM patients.

Upregulation of wild-type p53 by small molecule-induced elevation of NQO1 in non-small cell lung cancer cells.

The tumor suppressor p53 is usually inactivated by somatic mutations in malignant neoplasms, and its reactivation represents an attractive therapeutic strategy for cancers. Here, we reported that a new quinolone compound RYL-687 significantly inhibited non-small cell lung cancer (NSCLC) cells which express wild type (wt) p53, in contract to its much weaker cytotoxicity on cells with mutant p53. RYL-687 upregulated p53 in cells with wt but not mutant p53, and ectopic expression of wt p53 significantly enhanced the anti-NSCLC activity of this compound. RYL-687 induced production of reactive oxygen species (ROS) and upregulation of Nrf2, leading to an elevation of the NAD(P)H:quinoneoxidoreductase-1 (NQO1) that can protect p53 by inhibiting its degradation by 20S proteasome. RYL-687 bound NQO1, facilitating the physical interaction between NQO1 and p53. NQO1 was required for RYL-687-induced p53 accumulation, because silencing of NQO1 by specific siRNA or an NQO1 inhibitor uridine, drastically suppressed RYL-687-induced p53 upregulation. Moreover, a RYL-687-related prodrug significantly inhibited tumor growth in NOD-SCID mice inoculated with NSCLC cells and in a wt p53-NSCLC patient-derived xenograft mouse model. These data indicate that targeting NQO1 is a rational strategy to reactivate p53, and RYL-687 as a p53 stabilizer bears therapeutic potentials in NSCLCs with wt p53.

A novel PRRX1 loss-of-function variation contributing to familial atrial fibrillation and congenital patent ductus arteriosus.

Atrial fibrillation (AF) represents the most common type of sustained cardiac arrhythmia in humans and confers a significantly increased risk for thromboembolic stroke, congestive heart failure and premature death. Aggregating evidence emphasizes the predominant genetic defects underpinning AF and an increasing number of deleterious variations in more than 50 genes have been involved in the pathogenesis of AF. Nevertheless, the genetic basis underlying AF remains incompletely understood. In the current research, by whole-exome sequencing and Sanger sequencing analysis in a family with autosomal-dominant AF and congenital patent ductus arteriosus (PDA), a novel heterozygous variation in the PRRX1 gene encoding a homeobox transcription factor critical for cardiovascular development, NM_022716.4:c.373G>T;p.(Glu125*), was identified to be in co-segregation with AF and PDA in the whole family. The truncating variation was not detected in 306 unrelated healthy individuals employed as controls. Quantitative biological measurements with a reporter gene analysis system revealed that the Glu125*-mutant PRRX1 protein failed to transactivate its downstream target genes SHOX2 and ISL1, two genes that have been causally linked to AF. Conclusively, the present study firstly links PRRX1 loss-of-function variation to AF and PDA, suggesting that AF and PDA share a common abnormal developmental basis in a proportion of cases.

Detection and functional characterization of a novel MEF2A variation responsible for familial dilated cardiomyopathy.

OBJECTIVES: Dilated cardiomyopathy (DCM) represents the most frequent form of cardiomyopathy, leading to heart failure, cardiac arrhythmias and death. Accumulating evidence convincingly demonstrates the crucial role of genetic defects in the pathogenesis of DCM, and over 100 culprit genes have been implicated with DCM. However, DCM is of substantial genetic heterogeneity, and the genetic determinants underpinning DCM remain largely elusive. METHODS: Whole-exome sequencing and bioinformatical analyses were implemented in a consanguineous Chinese family with DCM. A total of 380 clinically annotated control individuals and 166 more DCM index cases then underwent Sanger sequencing analysis for the identified genetic variation. The functional characteristics of the variant were delineated by utilizing a dual-luciferase assay system. RESULTS: A heterozygous variation in the MEF2A gene (encoding myocyte enhancer factor 2A, a transcription factor pivotal for embryonic cardiogenesis and postnatal cardiac adaptation), NM_001365204.1: c.718G>T; p. (Gly240*), was identified, and verified by Sanger sequencing to segregate with autosome-dominant DCM in the family with complete penetrance. The nonsense variation was neither detected in 760 control chromosomes nor found in 166 more DCM probands. Functional analyses revealed that the variant lost transactivation on the validated target genes MYH6 and FHL2, both causally linked to DCM. Furthermore, the variation nullified the synergistic activation between MEF2A and GATA4, another key transcription factor involved in DCM. CONCLUSIONS: The findings firstly indicate that MEF2A loss-of-function variation predisposes to DCM in humans, providing novel insight into the molecular mechanisms of DCM and suggesting potential implications for genetic testing and prognostic evaluation of DCM patients.

SOX17 Loss-of-Function Mutation Underlying Familial Pulmonary Arterial Hypertension.

Pulmonary arterial hypertension (PAH) refers to a rare, progressive disorder that is characterized by occlusive pulmonary vascular remodeling, resulting in increased pulmonary arterial pressure, right-sided heart failure, and eventual death. Emerging evidence from genetic investigations of pediatric-onset PAH highlights the strong genetic basis underpinning PAH, and deleterious variants in multiple genes have been found to cause PAH. Nevertheless, PAH is of substantial genetic heterogeneity, and the genetic defects underlying PAH in the overwhelming majority of cases remain elusive. In this investigation, a consanguineous family suffering from PAH transmitted as an autosomal-dominant trait was identified. Through whole-exome sequencing and bioinformatic analyses as well as Sanger sequencing analyses of the PAH family, a novel heterozygous SOX17 mutation, NM_022454.4: c.379C>T; p. (Gln127*), was found to co-segregate with the disease in the family, with complete penetrance. The nonsense mutation was neither observed in 612 unrelated healthy volunteers nor retrieved in the population genetic databases encompassing the Genome Aggregation Database, the Exome Aggregation Consortium database, and the Single Nucleotide Polymorphism database. Biological analyses using a dual-luciferase reporter assay system revealed that the Gln127*-mutant SOX17 protein lost the ability to transcriptionally activate its target gene NOTCH1. Moreover, the Gln127*-mutant SOX17 protein exhibited no inhibitory effect on the function of CTNNB1-encode ß-catenin, which is a key player in vascular morphogenesis. This research firstly links SOX17 loss-of-function mutation to familial PAH, which provides novel insight into the molecular pathogenesis of PAH, suggesting potential implications for genetic and prognostic risk evaluation as well as personalized prophylaxis of the family members affected with PAH.

A New TBX5 Loss-of-Function Mutation Contributes to Congenital Heart Defect and Atrioventricular Block.

Congenital heart defect (CHD) represents the most common birth deformity, afflicting 1% of all births worldwide, and accounts for substantial morbidity and mortality. Increasing evidence highlights the pivotal roles of genetic etiologies in the pathogenesis of CHD, and pathogenic mutations in multiple genes, including TBX5 encoding a cardiac core transcription factor key to cardiovascular morphogenesis, have been involved in CHD. However, due to pronounced genetic heterogeneity of CHD, the genetic determinants underlying CHD in most cases remain obscure. In this investigation, by sequencing analysis of the coding exons and flanking introns of the TBX5 gene in 198 unrelated patients affected with CHD, a novel heterozygous mutation, NM_000192.3: c.692C>T; p. (Pro231Leu), was identified in an index patient with familial double outlet right ventricle (DORV), ventricular septal defect (VSD), and atrioventricular block (AVB). Genetic analysis of the proband's pedigree showed that the mutation co-segregated with the diseases. The missense mutation, which altered the amino acid conserved evolutionarily, was absent from 266 unrelated healthy subjects. Functional analyses with a dual-luciferase reporter assay system unveiled that the Pro231Leu-mutant TBX5 was associated with significantly reduced transcriptional activity on its target genes MYH6 and NPPA. Furthermore, the mutation disrupted the synergistic transactivation between TBX5 and NKX2-5 as well as GATA4, two other transcription factors causally linked to CHD. This study firstly links TBX5 loss-of-function mutation to familial DORV, VSD, and AVB, which provides novel insight into the mechanism underpinning CHD and AVB, suggesting potential implications for genetic evaluation and individualized treatment of patients affected by CHD and AVB.

A novel TBX5 mutation predisposes to familial cardiac septal defects and atrial fibrillation as well as bicuspid aortic valve.

TBX5 has been linked to Holt-Oram syndrome, with congenital heart defect (CHD) and atrial fibrillation (AF) being two major cardiac phenotypes. However, the prevalence of a TBX5 variation in patients with CHD and AF remains obscure. In this research, by sequencing analysis of TBX5 in 178 index patients with both CHD and AF, a novel heterozygous variation, NM_000192.3: c.577G>T; p.(Gly193*), was identified in one index patient with CHD and AF as well as bicuspid aortic valve (BAV), with an allele frequency of approximately 0.28%. Genetic analysis of the proband's pedigree showed that the variation co-segregated with the diseases. The pathogenic variation was not detected in 292 unrelated healthy subjects. Functional analysis by using a dual-luciferase reporter assay system showed that the Gly193*-mutant TBX5 protein failed to transcriptionally activate its target genes MYH6 and NPPA. Moreover, the mutation nullified the synergistic transactivation between TBX5 and GATA4 as well as NKX2-5. Additionally, whole-exome sequencing analysis showed no other genes contributing to the diseases. This investigation firstly links a pathogenic variant in the TBX5 gene to familial CHD and AF as well as BAV, suggesting that CHD and AF as well as BAV share a common developmental basis in a subset of patients.

ISL1 loss-of-function mutation contributes to congenital heart defects.

Congenital heart defect (CHD) is the most common form of birth deformity and is responsible for substantial morbidity and mortality in humans. Increasing evidence has convincingly demonstrated that genetic defects play a pivotal role in the pathogenesis of CHD. However, CHD is a genetically heterogeneous disorder and the genetic basis underpinning CHD in the vast majority of cases remains elusive. This study was sought to identify the pathogenic mutation in the ISL1 gene contributing to CHD. A cohort of 210 unrelated patients with CHD and a total of 256 unrelated healthy individuals used as controls were registered. The coding exons and splicing boundaries of ISL1 were sequenced in all study subjects. The functional effect of an identified ISL1 mutation was evaluated using a dual-luciferase reporter assay system. A novel heterozygous ISL1 mutation, c.409G > T or p.E137X, was identified in an index patient with congenital patent ductus arteriosus and ventricular septal defect. Analysis of the proband's pedigree revealed that the mutation co-segregated with CHD, which was transmitted in the family in an autosomal dominant pattern with complete penetrance. The nonsense mutation was absent in 512 control chromosomes. Functional analysis unveiled that the mutant ISL1 protein failed to transactivate the promoter of MEF2C, alone or in synergy with TBX20. This study firstly implicates ISL1 loss-of-function mutation with CHD in humans, which provides novel insight into the molecular mechanism of CHD, implying potential implications for genetic counseling and individually tailored treatment of CHD patients.

A New ISL1 Loss-of-Function Mutation Predisposes to Congenital Double Outlet Right Ventricle.

Occurring in about 1% of all live births, congenital heart defects (CHDs) represent the most frequent type of developmental abnormality and account for remarkably increased infant morbidity and mortality. Aggregating studies demonstrate that genetic components have a key role in the occurrence of CHDs. Nevertheless, due to pronounced genetic heterogeneity, the genetic causes of CHDs remain unclear in most patients. In this research, 114 unrelated patients affected with CHDs and 218 unrelated individuals without CHDs served as controls were recruited. The coding regions and splicing donors/acceptors of the ISL1 gene, which codes for a transcription factor required for proper cardiovascular development, were screened for mutations by sequencing in all study participants. The functional characteristics of an identified ISL1 mutation were delineated with a dual-luciferase reporter assay system. As a result, a new heterozygous ISL1 mutation, NM_002202.2: c.225C>G; p. (Tyr75*), was discovered in an index patient with double outlet right ventricle and ventricular septal defect. Analysis of the proband's family unveiled that the mutation co-segregated with the CHD phenotype. The nonsense mutation was absent in the 436 control chromosomes. Biological analysis showed that the mutant ISL1 protein had no transcriptional activity. Furthermore, the mutation nullified the synergistic activation between ISL1 and TBX20, another CHD-associated transcription factor. This research for the first time links an ISL1 loss-of-function mutation to double outlet right ventricle in humans, which adds insight to the molecular pathogenesis underpinning CHDs, suggesting potential implications for timely personalized management of CHD patients.

MEF2C loss-of-function mutation associated with familial dilated cardiomyopathy.

BACKGROUND: The MADS-box transcription factor myocyte enhancer factor 2C (MEF2C) is required for the cardiac development and postnatal adaptation and in mice-targeted disruption of the MEF2C gene results in dilated cardiomyopathy (DCM). However, in humans, the association of MEF2C variation with DCM remains to be investigated. METHODS: The coding regions and splicing boundaries of the MEF2C gene were sequenced in 172 unrelated patients with idiopathic DCM. The available close relatives of the index patient harboring an identified MEF2C mutation and 300 unrelated, ethnically matched healthy individuals used as controls were genotyped for MEF2C. The functional effect of the mutant MEF2C protein was characterized in contrast to its wild-type counterpart by using a dual-luciferase reporter assay system. RESULTS: A novel heterozygous MEF2C mutation, p.Y157X, was detected in an index patient with adult-onset DCM. Genetic screen of the mutation carrier's family members revealed that the mutation co-segregated with DCM, which was transmitted as an autosomal dominant trait with complete penetrance. The non-sense mutation was absent in 300 control individuals. Functional analyses unveiled that the mutant MEF2C protein had no transcriptional activity. Furthermore, the mutation abolished the synergistic transactivation between MEF2C and GATA4 as well as HAND1, two other transcription factors that have been associated with DCM. CONCLUSIONS: This study indicates MEF2C as a new gene responsible for human DCM, which provides novel insight into the mechanism underpinning DCM, suggesting potential implications for development of innovative prophylactic and therapeutic strategies for DCM, the most prevalent form of primary myocardial disease.

A SHOX2 loss-of-function mutation underlying familial atrial fibrillation.

Atrial fibrillation (AF), as the most common sustained cardiac arrhythmia, is associated with substantially increased morbidity and mortality. Aggregating evidence demonstrates that genetic defects play a crucial role in the pathogenesis of AF, especially in familial AF. Nevertheless, AF is of pronounced genetic heterogeneity, and in an overwhelming majority of cases the genetic determinants underlying AF remain elusive. In the current study, 162 unrelated patients with familial AF and 238 unrelated healthy individuals served as controls were recruited. The coding exons and splicing junction sites of the SHOX2 gene, which encodes a homeobox-containing transcription factor essential for proper development and function of the cardiac conduction system, were sequenced in all study participants. The functional effect of the mutant SHOX2 protein was characterized with a dual-luciferase reporter assay system. As a result, a novel heterozygous SHOX2 mutation, c.580C>T or p.R194X, was identified in an index patient, which was absent from the 476 control chromosomes. Genetic analysis of the proband's pedigree revealed that the nonsense mutation co-segregated with AF in the family with complete penetrance. Functional assays demonstrated that the mutant SHOX2 protein had no transcriptional activity compared with its wild-type counterpart. In conclusion, this is the first report on the association of SHOX2 loss-of-function mutation with enhanced susceptibility to familial AF, which provides novel insight into the molecular mechanism underpinning AF, suggesting potential implications for genetic counseling and individualized management of AF patients.

ZBTB17 loss-of-function mutation contributes to familial dilated cardiomyopathy.

Dilated cardiomyopathy (DCM) is a common primary myocardial disease leading to congestive heart failure, arrhythmia and sudden cardiac death. Increasing studies demonstrate substantial genetic determinants for DCM. Nevertheless, DCM is of substantial genetic heterogeneity, and the genetic basis for DCM in most patients remains unclear. The present study was sought to investigate the association of a genetic variant in the ZBTB17 gene with DCM. A cohort of 158 unrelated patients with idiopathic DCM and a total of 230 unrelated, ethnically matched healthy individuals used as controls were recruited. The coding exons and splicing boundaries of ZBTB17 were sequenced in all study participants. The functional effect of the mutant ZBTB17 was characterized by a dual-luciferase reporter assay system. A novel heterozygous ZBTB17 mutation, p.E243X, was discovered in an index patient. Genetic scan of the mutation carrier's available relatives showed that the mutation was present in all affected family members but absent in unaffected family members. Analysis of the proband's pedigree revealed that the mutation co-segregated with DCM, which was transmitted in an autosomal dominant pattern with complete penetrance. The nonsense mutation was absent in the 460 control chromosomes. Functional assays demonstrated that the truncated ZBTB17 protein had no transcriptional activity as compared with its wild-type counterpart. This study firstly associates ZBTB17 loss-of-function mutation with enhanced susceptibility to DCM in humans, which provides novel insight into the molecular mechanism underpinning DCM, implying potential implications for genetic counseling and personalized management of DCM.

A Novel MEF2C Loss-of-Function Mutation Associated with Congenital Double Outlet Right Ventricle.

Congenital heart defect (CHD) represents the most prevalent birth defect, and accounts for substantial morbidity and mortality in humans. Aggregating evidence demonstrates the genetic basis for CHD. However, CHD is a heterogeneous disease, and the genetic determinants underlying CHD in most patients remain unknown. In the present study, a cohort of 186 unrelated cases with CHD and 300 unrelated control individuals were recruited. The coding exons and flanking introns of the MEF2C gene, which encodes a transcription factor crucial for proper cardiovascular development, were sequenced in all study participants. The functional effect of an identified MEF2C mutation was characterized using a dual-luciferase reporter assay system. As a result, a novel heterozygous MEF2C mutation, p.R15C, was detected in an index patient with congenital double outlet right ventricle (DORV) as well as ventricular septal defect. Analysis of the proband's pedigree showed that the mutation co-segregated with CHD with complete penetrance. The missense mutation, which changed the evolutionarily conserved amino acid, was absent in 300 control individuals. Functional deciphers revealed that the mutant MEF2C protein had a significantly decreased transcriptional activity. Furthermore, the mutation significantly reduced the synergistic activation between MEF2C and GATA4, another transcription factor linked to CHD. This study firstly associates MEF2C loss-of-function mutation with DORV in humans, which provides novel insight into the molecular pathogenesis of CHD, suggesting potential implications for genetic counseling and personalized treatment of CHD patients.

CASZ1 loss-of-function mutation contributes to familial dilated cardiomyopathy.

BACKGROUND: The zinc finger transcription factor CASZ1 plays a key role in cardiac development and postnatal adaptation, and in mice, deletion of the CASZ1 gene leads to dilated cardiomyopathy (DCM). However, in humans whether genetically defective CASZ1 contributes to DCM remains unclear. METHODS: The coding exons and splicing junction sites of the CASZ1 gene were sequenced in 138 unrelated patients with idiopathic DCM. The available family members of the index patient harboring an identified CASZ1 mutation and 200 unrelated, ethnically matched healthy individuals used as controls were genotyped for CASZ1. The functional characteristics of the mutant CASZ1 were analyzed in contrast to its wild-type counterpart using a luciferase reporter assay system. RESULTS: A novel heterozygous CASZ1 mutation, p.K351X, was identified in an index patient with DCM. Genetic analysis of the mutation carrier's family showed that the mutation co-segregated with DCM, which was transmitted in an autosomal dominant pattern with complete penetrance. The nonsense mutation, which was absent in 400 referential chromosomes, altered the amino acid that was highly conserved evolutionarily. Biological investigations revealed that the mutant CASZ1 had no transcriptional activity. CONCLUSIONS: The current study reveals CASZ1 as a new gene responsible for human DCM, which provides novel mechanistic insight and potential therapeutic target for CASZ1-associated DCM, implying potential implications in improved prophylactic and therapeutic strategies for DCM, the most common type of primary myocardial disease.

MEF2C loss-of-function mutation contributes to congenital heart defects.

Congenital heart disease (CHD) is the most common type of developmental abnormality in humans, and is a leading cause for substantially increased morbidity and mortality in affected individuals. Increasing studies demonstrates a pivotal role of genetic defects in the pathogenesis of CHD, and presently mutations in more than 60 genes have been associated with CHD. Nevertheless, CHD is of pronounced genetic heterogeneity, and the genetic basis underpinning CHD in a large proportion of patients remains unclear. In the present study, the whole coding exons and splicing donors/acceptors of the MEF2C gene, which codes for a transcription factor essential for normal cardiovascular development, were sequenced in 200 unrelated patients affected with CHD, and a novel heterozygous missense mutation, p.L38P, was identified in an index patient with patent ductus arteriosus (PDA) and ventricular septal defect (VSD). Genetic scan of the mutation carrier's family members available showed that the mutation was present in all affected family members but absent in unaffected family members. Analysis of the proband's pedigree revealed that the mutation co-segregated with PDA, which was transmitted as an autosomal dominant trait with complete penetrance. The mutation changed the amino acid that was completely conserved evolutionarily, and did not exist in 300 unrelated, ethnically-matched healthy individuals used as controls. Functional deciphers by using a dual-luciferase reporter assay system unveiled that the mutant MEF2C protein had a significantly reduced transcriptional activity. Furthermore, the mutation significantly diminished the synergistic activation between MEF2C and GATA4, another cardiac core transcription factor that has been causally linked to CHD. In conclusion, this is the first report on the association of a MEF2C loss-of-function mutation with an increased vulnerability to CHD in humans, which provides novel insight into the molecular mechanisms underlying CHD, implying potential implications for early diagnosis and timely prophylaxis of CHD.

TBX20 loss-of-function mutation responsible for familial tetralogy of Fallot or sporadic persistent truncus arteriosus.

Congenital heart disease (CHD), the most common form of developmental abnormality in humans, remains a leading cause of morbidity and mortality in neonates. Genetic defects have been recognized as the predominant causes of CHD. Nevertheless, CHD is of substantial genetic heterogeneity and the genetic defects underlying CHD in most cases remain unclear. In the current study, the coding regions and splicing junction sites of the TBX20 gene, which encodes a T-box transcription factor key to cardiovascular morphogenesis, were sequenced in 175 unrelated patients with CHD, and a novel heterozygous TBX20 mutation, p.K274X, was identified in an index patient with tetralogy of Fallot (TOF). Genetic analysis of the proband's available family members showed that his father, elder brother and son had also TOF. In addition, his father and elder brother had also atrial septal defect, and his niece had persistent truncus arteriosus and ventricular septal defect. Analysis of the pedigree revealed that the mutation co-segregated with CHD transmitted in an autosomal dominant fashion, with complete penetrance. The nonsense mutation, which was absent in the 800 control chromosomes, was predicted to produce a truncated protein with only the amino terminus and partial T-box domain left. Functional analyses by using a dual-luciferase reporter assay system showed that the mutant TBX20 lost the ability to transactivate the target gene ANF. Furthermore, the mutation reduced the synergistic activation between TBX20 and NKX2.5 as well as GATA4, two other transcriptional factors previously associated with various CHD, encompassing TOF. This study firstly links TBX20 loss-of-function mutation to familial TOF or sporadic persistent truncus arteriosus, providing novel insight into the molecular pathogenesis of CHD.

HAND1 Loss-of-Function Mutation Causes Tetralogy of Fallot.

As the most prevalent form of birth defect in humans worldwide, congenital heart disease (CHD) is responsible for substantial morbidity and is still the leading cause of birth defect-related demises. Increasing evidence demonstrates that genetic defects play an important role in the pathogenesis of CHD, and mutations in multiple genes, especially in those coding for cardiac core transcription factors, have been causally linked to various CHDs. Nevertheless, CHD is a genetically heterogeneous disease and the genetic determinants underpinning CHD in an overwhelming majority of patients remain elusive. In the current study, genomic DNA was extracted from venous blood samples of 165 unrelated patients with CHD, and the coding exons and splicing junction sites of the HAND1 gene, which encodes a basic helix-loop-helix transcription factor essential for cardiovascular development, were sequenced. As a result, a novel heterozygous mutation, p.R118C, was identified in a patient with tetralogy of Fallot (TOF). The missense mutation, which was absent in 600 referential chromosomes, altered the amino acid that was completely conserved evolutionarily. Biological assays with a dual-luciferase reporter assay system revealed that the R118C-mutant HAND1 protein had significantly reduced transcriptional activity when compared with its wild-type counterpart. Furthermore, the mutation significantly decreased the synergistic activation of a downstream target gene between HAND1 and GATA4, another cardiac core transcription factor associated with TOF. To our knowledge, this is the first report on the association of a HAND1 loss-of-function mutation with enhanced susceptibility to TOF in humans. The findings provide novel insight into the molecular etiology underlying TOF, suggesting potential implications for the improved prophylactic and therapeutic strategies for TOF.

Prevalence and Spectrum of NKX2-5 Mutations Associated With Sporadic Adult-Onset Dilated Cardiomyopathy.

Dilated cardiomyopathy (DCM), the most common form of primary myocardial disease, is a leading cause of congestive heart failure and the most common indication for heart transplantation. Recently, NKX2-5 mutations have been involved in the pathogenesis of familial DCM. However, the prevalence and spectrum of NKX2-5 mutations associated with sporadic DCM remain to be evaluated. In this study, the coding regions and flanking introns of the NKX2-5 gene, which encodes a cardiac transcription factor pivotal for cardiac development and structural remodeling, were sequenced in 210 unrelated patients with sporadic adult-onset DCM. A total of 300 unrelated healthy individuals used as controls were also genotyped for NKX2-5. The functional effect of the mutant NKX2-5 was investigated using a dual-luciferase reporter assay system. As a result, two novel heterozygous NKX2-5 mutations, p.R139W and p.E167X, were identified in 2 unrelated patients with sporadic adult-onset DCM, with a mutational prevalence of approximately 0.95%. The mutations were absent in 600 referential chromosomes and the altered amino acids were completely conserved evolutionarily across species. Functional assays revealed that the NKX2-5 mutants were associated with significantly reduced transcriptional activity. Furthermore, the mutations abrogated the synergistic activation between NKX2-5 and GATA4 as well as TBX20, two other cardiac key transcription factors that have been causally linked to adult-onset DCM. This study is the first to associate NKX2-5 loss-of-function mutations with enhanced susceptibility to sporadic DCM, which provides novel insight into the molecular etiology underpinning DCM, and suggests the potential implications for the genetic counseling and personalized treatment of the DCM patients.

Increased Expression of Caspase-12 After Experimental Subarachnoid Hemorrhage.

Convincing evidences have proved that apoptosis plays a vital role in the pathogenesis of early and delayed brain injury following subarachnoid hemorrhage (SAH). Recently, a novel caspase-12-mediated apoptotic pathway has been reported to be induced by excess endoplasmic reticulum (ER) stress. Extensive protein damage occurs after SAH, which may trigger ER stress-associated apoptotic pathway. Thus, we hypothesized that caspase-12, as the major molecular marker of this novel apoptotic pathway, may be activated and involved in the pathogenesis of apoptotic injury after SAH. This study sought to investigate the changes of caspase-12 expressions in both in vitro and in vivo SAH models. Western blot analysis found significantly increased protein expressions of both pro- and active forms of caspase-12 after SAH. Quantitative real-time PCR and immunohistochemistry assays confirmed elevated caspase-12 level after SAH in vivo. Further, double immunofluorescence staining revealed obvious caspase-12 over-expression in both cortical neurons and astrocytes. Moreover, immunofluorescent co-staining in vivo demonstrated that neural cells with high immunoreactivity of caspase-12 also expressed caspase-3, and dual-immunofluorescent staining for caspase-12 and TUNEL in vitro showed that TUNEL-positive cells were more likely to exhibit higher caspase-12 immunoreactivity, indicating a potential contribution of caspase-12 activation to apoptosis in SAH. Collectively, our results showed significant upregulation of caspase-12 expression after experimental SAH. These findings also offer important implications for further investigations of the therapeutic potential of caspase-12 associated apoptosis in SAH.

TBX20 loss-of-function mutation associated with familial dilated cardiomyopathy.

BACKGROUND: Dilated cardiomyopathy (DCM) is a major cause of congestive heart failure, sudden cardiac death and cardiac transplantation. Aggregating evidence highlights the genetic origin of DCM. However, DCM is a genetically heterogeneous disorder, and the genetic components underlying DCM in most cases remain unknown. METHODS: The coding regions and splicing junction sites of the TBX20 gene were sequenced in 120 unrelated patients with idiopathic DCM. The available close relatives of the index patient carrying an identified mutation and 300 unrelated ethnically matched healthy individuals used as controls were genotyped for TBX20. The functional characteristics of the mutant TBX20 were assayed in contrast to its wild-type counterpart by using a dual-luciferase reporter assay system. RESULTS: A novel heterozygous TBX20 mutation, p.F256I, was identified in a family with DCM transmitted in an autosomal dominant fashion, which co-segregated with DCM in the family with complete penetrance. The missense mutation was absent in 600 control chromosomes and the altered amino acid was completely conserved evolutionarily among various species. Functional assays revealed that the mutant TBX20 had significantly diminished transcriptional activity. Furthermore, the mutation markedly reduced the synergistic activation of TBX20 with NKX2-5 or GATA4. CONCLUSIONS: This study links TBX20 loss-of-function mutation to idiopathic DCM in humans for the first time, providing novel insight into the molecular mechanism underpinning DCM.