Chaga Medicinal Mushroom, Inonotus obliquus (Agaricomycetes), Polysaccharides Alleviate Photoaging by Regulating Nrf2 Pathway and Autophagy.

Inonotus obliquus is a medicinal mushroom that contains the valuable I. obliquus polysaccharides (IOP), which is known for its bioactive properties. Studies have shown that IOP could inhibit oxidative stress induced premature aging and DNA damage, and delay body aging. However, the molecular mechanism of IOP in improving skin photoaging remains unclear, which prevents the development and utilization of I. obliquus in the field of skin care. In this study, ultraviolet B (UVB) induced human immortalized keratinocyte (HaCaT) cell photoaging model was used to explore the mechanism of IOP in relieving skin photoaging. Results showed that IOP inhibited cell senescence and apoptosis by reducing the protein expressions of p16, p21, and p53. IOP increased HO-1, SOD, and CAT expressions to achieve Nrf2/HO-1 pathway, thus improving antioxidant effects and preventing ROS generation. Furthermore, IOP enhanced the expression levels of p-AMPK, LC3B, and Beclin-1 to alleviate the autophagy inhibition in UVB-induced HaCaT cells. Based on these findings, our data suggested that IOP may be used to develop effective natural anti-photoaging ingredients to promote skin health.

Centella asiatica alleviates psoriasis through JAK/STAT3-mediated inflammation: An in vitro and in vivo study.

ETHNOPHARMACOLOGICAL RELEVANCE: Centella asiatica (L.) Urban (CA) is a dry herb of the Umbelliferae family, first mentioned in Shennong's Herbal Classic. It is known for its ability to clear heat and dampness, detoxify, and reduce swelling, making it a popular treatment for dermatitis, wound healing, and lupus erythematosus. Psoriasis is a chronic inflammatory skin disease that is characterized by clearly delineated erythema and squamous skin lesions. However, the effect of CA on regulating inflammation and its mechanism in the pathogenesis of psoriasis is still not fully understood. AIM OF THE STUDY: This study evaluated the effects of CA on inflammatory dermatosis by in vitro and in vivo studies. And clarified the important role of the JAK/STAT3 signaling pathway in the treatment of psoriasis with CA. METHODS AND MATERIALS: Different components of CA were extracted and analyzed for their total flavonoid and polyphenol contents. The antioxidant capacity of the CA extracts was determined using DPPH, ABTS, and FRAP methods. In vitro, HaCaT cells were induced by lipopolysaccharide (LPS, 20 µg·mL-1) to establish an inflammatory injury model, and the effects of CA extracts on oxidative stress, inflammation and skin barrier function were evaluated systematically. Annexin V-FITC/PI staining was utilized for detecting cell apoptosis, while the expression of NF-κB and JAK/STAT3 pathways were detected by RT-PCR and western blot. Combined with an in vivo mice model of Imiquimod (IMQ) induced psoriasis-like skin inflammation, the most effective CA extract for alleviating psoriasis was identified and its potential mechanism was investigated. RESULTS: CA extracts showed high antioxidant capacity and were able to increase the content of GSH and SOD while reducing intracellular ROS generation. Notably, CA ethyl acetate extract (CAE) was found to be the most effective. Furthermore, CA extracts effectively downregulate inflammatory factors (IFN-γ, CCL20, IL-6 and TNF-α) mRNA levels and improved the gene expressions of barrier protective factors AQP3 and FLG, among them CAE and n-hexane extract of CA (CAH) had better effects. Western blot analysis indicated that CAE and CAH had anti-inflammatory effects by inhibiting the activation of NF-κB and JAK/STAT3 pathways, and CAE exhibited the best regulatory effect at the dose of 25 µg·mL-1. In vivo experiment, the psoriasis-like skin inflammation mice model was established by 5% IMQ and treated CAE solution (10, 20, 40 mg·mL-1) for 7 days, the results showed that CAE intervention reduced the skin scale and blood scab, and significantly inhibited the secretion of inflammatory factors in both serum and skin lesions at the dose of 40 mg·mL-1. CONCLUSION: Centella asiatica extracts were effective in improving skin inflammation and skin barrier dysfunction, and also alleviated psoriasis through JAK/STAT3 pathway. The results provided experimental support for the potential use of Centella asiatica in functional food and skin care products.

VEZF1 loss-of-function mutation underlying familial dilated cardiomyopathy.

Dilated cardiomyopathy (DCM), characteristic of left ventricular or biventricular dilation with systolic dysfunction, is the most common form of cardiomyopathy, and a leading cause of heart failure and sudden cardiac death. Aggregating evidence highlights the underlying genetic basis of DCM, and mutations in over 100 genes have been causally linked to DCM. Nevertheless, due to pronounced genetic heterogeneity, the genetic defects underpinning DCM in most cases remain obscure. Hence, this study was sought to identify novel genetic determinants of DCM. In this investigation, whole-exome sequencing and bioinformatics analyses were conducted in a family suffering from DCM, and a novel heterozygous mutation in the VEZF1 gene (coding for a zinc finger-containing transcription factor critical for cardiovascular development and structural remodeling), NM_007146.3: c.490A > T; p.(Lys164*), was identified. The nonsense mutation was validated by Sanger sequencing and segregated with autosome-dominant DCM in the family with complete penetrance. The mutation was neither detected in another cohort of 200 unrelated DCM patients nor observed in 400 unrelated healthy individuals nor retrieved in the Single Nucleotide Polymorphism database, the Human Gene Mutation Database and the Genome Aggregation Database. Biological analyses by utilizing a dual-luciferase reporter assay system revealed that the mutant VEZF1 protein failed to transactivate the promoters of MYH7 and ET1, two genes that have been associated with DCM. The findings indicate VEZF1 as a new gene responsible for DCM, which provides novel insight into the molecular pathogenesis of DCM, implying potential implications for personalized precisive medical management of the patients affected with DCM.

Identification of SOX18 as a New Gene Predisposing to Congenital Heart Disease.

Congenital heart disease (CHD) is the most frequent kind of birth deformity in human beings and the leading cause of neonatal mortality worldwide. Although genetic etiologies encompassing aneuploidy, copy number variations, and mutations in over 100 genes have been uncovered to be involved in the pathogenesis of CHD, the genetic components predisposing to CHD in most cases remain unclear. We recruited a family with CHD from the Chinese Han population in the present investigation. Through whole-exome sequencing analysis of selected family members, a new SOX18 variation, namely NM_018419.3:c.349A>T; p.(Lys117*), was identified and confirmed to co-segregate with the CHD phenotype in the entire family by Sanger sequencing analysis. The heterozygous variant was absent from the 384 healthy volunteers enlisted as control individuals. Functional exploration via luciferase reporter analysis in cultivated HeLa cells revealed that Lys117*-mutant SOX18 lost transactivation on its target genes NR2F2 and GATA4, two genes responsible for CHD. Moreover, the genetic variation terminated the synergistic activation between SOX18 and NKX2.5, another gene accountable for CHD. The findings strongly indicate SOX18 as a novel gene contributing to CHD, which helps address challenges in the clinical genetic diagnosis and prenatal prophylaxis of CHD.

SOX7 loss-of-function variation as a cause of familial congenital heart disease.

INTRODUCTION: As the most frequent type of birth defect in humans, congenital heart disease (CHD) leads to a large amount of morbidity and mortality as well as a tremendous socioeconomic burden. Accumulating studies have convincingly substantiated the pivotal roles of genetic defects in the occurrence of familial CHD, and deleterious variations in a great number of genes have been reported to cause various types of CHD. However, owing to pronounced genetic heterogeneity, the hereditary components underpinning CHD remain obscure in most cases. This investigation aimed to identify novel genetic determinants underlying CHD. METHODS AND RESULTS: A four-generation pedigree with high incidence of autosomal-dominant CHD was enrolled from the Chinese Han race population. Using whole-exome sequencing and Sanger sequencing assays of the family members available, a novel SOX7 variation in heterozygous status, NM_031439.4: c.310C>T; p.(Gln104*), was discovered to be in co-segregation with the CHD phenotype in the whole family. The truncating variant was absent in 500 unrelated healthy subjects utilized as control individuals. Functional measurements by dual-luciferase reporter analysis revealed that Gln104*-mutant SOX7 failed to transactivate its two important target genes, GATA4 and BMP2, which are both responsible for CHD. In addition, the nonsense variation invalidated the cooperative transactivation between SOX7 and NKX2.5, which is another recognized CHD-causative gene. CONCLUSION: The present study demonstrates for the first time that genetically defective SOX7 predisposes to CHD, which sheds light on the novel molecular mechanism underpinning CHD, and implies significance for precise prevention and personalized treatment in a subset of CHD patients.

KLF15 Loss-of-Function Mutation Underlying Atrial Fibrillation as well as Ventricular Arrhythmias and Cardiomyopathy.

Atrial fibrillation (AF) represents the most common type of clinical cardiac arrhythmia and substantially increases the risks of cerebral stroke, heart failure and death. Accumulating evidence has convincingly demonstrated the strong genetic basis of AF, and an increasing number of pathogenic variations in over 50 genes have been causally linked to AF. Nevertheless, AF is of pronounced genetic heterogeneity, and the genetic determinants underpinning AF in most patients remain obscure. In the current investigation, a Chinese pedigree with AF as well as ventricular arrhythmias and hypertrophic cardiomyopathy was recruited. Whole exome sequencing and bioinformatic analysis of the available family members were conducted, and a novel heterozygous variation in the KLF15 gene (encoding Krüppel-like factor 15, a transcription factor critical for cardiac electrophysiology and structural remodeling), NM_014079.4: c.685A>T; p.(Lys229*), was identified. The variation was verified by Sanger sequencing and segregated with autosomal dominant AF in the family with complete penetrance. The variation was absent from 300 unrelated healthy subjects used as controls. In functional assays using a dual-luciferase assay system, mutant KLF15 showed neither transcriptional activation of the KChIP2 promoter nor transcriptional inhibition of the CTGF promoter, alone or in the presence of TGFB1, a key player in the pathogenesis of arrhythmias and cardiomyopathies. The findings indicate KLF15 as a new causative gene responsible for AF as well as ventricular arrhythmias and hypertrophic cardiomyopathy, and they provide novel insight into the molecular mechanisms underlying cardiac arrhythmias and hypertrophic cardiomyopathy.

SOX17 loss-of-function variation underlying familial congenital heart disease.

As the most prevalent form of human birth defect, congenital heart disease (CHD) contributes to substantial morbidity, mortality and socioeconomic burden worldwide. Aggregating evidence has convincingly demonstrated that genetic defects exert a pivotal role in the pathogenesis of CHD, and causative mutations in multiple genes have been causally linked to CHD. Nevertheless, CHD is of pronounced genetic heterogeneity, and the genetic components underpinning CHD in the overwhelming majority of patients remain obscure. In this research, a four-generation consanguineous family suffering from CHD transmitted in an autosomal dominant mode was recruited. By whole-exome sequencing and bioinformatics analyses as well as Sanger sequencing analyses of the family members, a new heterozygous SOX17 variation, NM_022454.4: c.553G > T; p.(Glu185*), was identified to co-segregate with CHD in the family, with complete penetrance. The nonsense variation was neither detected in 310 unrelated healthy volunteers used as controls nor retrieved in such population genetics databases as the Exome Aggregation Consortium database, Genome Aggregation Database, and the Single Nucleotide Polymorphism database. Functional assays by utilizing a dual-luciferase reporter assay system unveiled that the Glu185*-mutant SOX17 protein had no transcriptional activity on its two target genes NOTCH1 and GATA4, which have been reported to cause CHD. Furthermore, the mutation abrogated the synergistic transactivation between SOX17 and NKX2.5, another established CHD-causing transcription factor. These findings firstly indicate SOX17 loss-of-function mutation predisposes to familial CHD, which adds novel insight to the molecular mechanism of CHD, implying potential implications for genetic risk appraisal and individualized prophylaxis of the family members affected with CHD.

miR-496/MMP10 Is Involved in the Proliferation of IL-1ß-Induced Fibroblast-Like Synoviocytes Via Mediating the NF-κB Signaling Pathway.

Rheumatoid arthritis (RA) is a common chronic autoimmune disease featured by synovial inflammation. miR-496 is closely involved in various pathologic conditions. However, its role in RA has not yet been elucidated. Expression of miR-496 and MMP10 was determined based on the clinical samples with RA retrieved from the Gene Expression Omnibus (GEO) datasets. In vitro model of RA was constructed in MH7A cells stimulated by IL-1ß (10 ng/mL). Cell counting kit 8 (CCK-8) and flow cytometry experiments were implemented to investigate the cell viability and apoptosis rate of MH7A cells. TargetScan was applied to identify the targets of miR-496, and the regulation of miR-496 on MMP10 expression was validated by a dual-luciferase reporter gene assay. qRT-PCR and western blot analyses were conducted to examine the expression. miR-496 expression was decreased in RA tissues and MH7A cells after IL-1ß treatment. Overexpression of miR-496 significantly inhibited IL-1ß-treated MH7A cell viability. MMP10 was identified as a target of miR-496 and its expression was negatively regulated by miR-496. The effects of miR-496 on MH7A cell proliferation and apoptosis were reversed by MMP10. The activity of NF-κB pathway was associated with the miR-496/MMP10 axis in IL-1ß-stimulated MH7A cells. To summarize, this study demonstrated that miR-496 can impair the proliferative ability and facilitate the apoptosis of IL-1ß-treated MH7A through regulating MMP10 expression and NF-κB signaling pathway.

An ultra-broadband terahertz metamaterial coherent absorber using multilayer electric ring resonator structures based on anti-reflection coating.

We propose a method for achieving THz ultra-broadband coherent absorption using the anti-reflection theory of metamaterials. The metamaterial absorber consists of a periodic array of electric ring resonators with a multilayered structure which form the desired refractive index dispersion and provide continuous anti-reflection over a wide frequency range. The destructive interference mechanism and resonance absorption of the absorber are determined by simulation analysis and numerical simulation. Simulation results show that the absorption bandwidth is almost 8.02 THz (absorption rate >90%) over the entire terahertz band (0.1 THz-10 THz). This design provides an effective and viable method for constructing broadband absorbers for stealth technology and the construction of enhanced transmittance devices.

MicroRNA changes of bone marrow-derived mesenchymal stem cells differentiated into neuronal-like cells by Schwann cell-conditioned medium.

Bone marrow-derived mesenchymal stem cells differentiate into neurons under the induction of Schwann cells. However, key microRNAs and related pathways for differentiation remain unclear. This study screened and identified differentially expressed microRNAs in bone marrow-derived mesenchymal stem cells induced by Schwann cell-conditioned medium, and explored targets and related pathways involved in their differentiation into neuronal-like cells. Primary bone marrow-derived mesenchymal stem cells were isolated from femoral and tibial bones, while primary Schwann cells were isolated from bilateral saphenous nerves. Bone marrow-derived mesenchymal stem cells were cultured in unconditioned (control group) and Schwann cell-conditioned medium (bone marrow-derived mesenchymal stem cell + Schwann cell group). Neuronal differentiation of bone marrow-derived mesenchymal stem cells induced by Schwann cell-conditioned medium was observed by time-lapse imaging. Upon induction, the morphology of bone marrow-derived mesenchymal stem cells changed into a neural shape with neurites. Results of quantitative reverse transcription-polymerase chain reaction revealed that nestin mRNA expression was upregulated from 1 to 3 days and downregulated from 3 to 7 days in the bone marrow-derived mesenchymal stem cell + Schwann cell group. Compared with the control group, microtubule-associated protein 2 mRNA expression gradually increased from 1 to 7 days in the bone marrow-derived mesenchymal stem cell + Schwann cell group. After 7 days of induction, microRNA analysis identified 83 significantly differentially expressed microRNAs between the two groups. Gene Ontology analysis indicated enrichment of microRNA target genes for neuronal projection development, regulation of axonogenesis, and positive regulation of cell proliferation. Kyoto Encyclopedia of Genes and Genomes pathway analysis demonstrated that Hippo, Wnt, transforming growth factor-beta, and Hedgehog signaling pathways were potentially associated with neural differentiation of bone marrow-derived mesenchymal stem cells. This study, which carried out successful microRNA analysis of neuronal-like cells differentiated from bone marrow-derived mesenchymal stem cells by Schwann cell induction, revealed key microRNAs and pathways involved in neural differentiation of bone marrow-derived mesenchymal stem cells. All protocols were approved by the Animal Ethics Committee of Institute of Radiation Medicine, Chinese Academy of Medical Sciences on March 12, 2017 (approval number: DWLI-20170311).

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.

GATA4 Loss-of-Function Mutation and the Congenitally Bicuspid Aortic Valve.

Aggregating evidence suggests that genetic determinants play a pivotal role in the pathogenesis of the congenitally bicuspid aortic valve (BAV). BAV is of pronounced genetic heterogeneity, and the genetic components underlying BAV in an overwhelming majority of patients remain elusive. In the current study, the whole coding exons and adjacent introns, as well as 5' and 3' untranslated regions of the GATA4 gene, which codes for a zinc-finger transcription factor crucial for the normal development of the aortic valve, were screened by direct sequencing in 150 index patients with congenital BAV. The available family members of an identified mutation carrier and 300 unrelated, ethnically matched healthy individuals used as controls were also genotyped for GATA4. The functional effect of the mutation was characterized using a dual-luciferase reporter assay system. As a result, a novel heterozygous GATA4 mutation, p.E147X, was identified in a family with BAV transmitted in an autosomal dominant pattern. The nonsense mutation was absent in 600 control chromosomes. Functional deciphers revealed that the mutant GATA4 protein lost transcriptional activity compared with its wild-type counterpart. Furthermore, the mutation disrupted the synergistic transcriptional activation between GATA4 and NKX2.5, another transcription factor responsible for BAV. In conclusion, this study associates the GATA4 loss-of-function mutation with enhanced susceptibility to a BAV, thus providing novel insight into the molecular mechanism underpinning the BAV.

Nafamostat mesilate attenuates inflammation and apoptosis and promotes locomotor recovery after spinal cord injury.

AIM: Spinal cord injury (SCI) leads to severe neural damage for which there is currently no effective treatment. Exploration of the neuroprotective effect among clinically approved drugs will speed up clinical translation of SCI. Nafamostat mesilate (NM) as a synthetic serine protease inhibitor has been used clinically in pancreatitis treatments. However, its effectiveness in SCI is unknown. The aim of this study was to confirm the efficacy of NM in ameliorating SCI. METHODS: Intraperitoneal administration of NM was performed on a contusion SCI model in Wistar rat. Hematoxylin and eosin staining (H&E staining) and Luxol fast blue (LFB) staining were used to observe the histological lesions. Apoptosis was examined by TUNEL staining, Annexin V-FITC/PI, caspase-3, and Bcl-2. Cytokines and neurotrophins were tested by Western blot. Locomotion recovery assessed by hindlimb BBB score and the inclined plane test. RESULTS: Nafamostat mesilate treatment significantly improved locomotion recovery as assessed by hindlimb BBB scores and the inclined plane test. H&E staining and LFB staining showed a significant increase in spared tissue in both gray matter and white matter. NM decreased the expression of the proinflammatory cytokines TNF-α and IL-6. In addition, apoptosis was also significantly decreased, as shown by TUNEL staining and Annexin V-FITC/PI and by Western blotting for caspase-3 and Bcl-2 expression. Due to the mechanism of action of NM as a serine protease inhibitor, the drug decreased thrombin expression in the damaged spinal cord. Furthermore, NM increased the expression of neurotrophins (NT-3, BDNF, and NGF). CONCLUSIONS: Upon NM treatment, the functional and histological outcomes were improved, and microenvironment upon SCI was modulated. As a clinically approved drug, NM holds promise for clinical use after spinal cord injury.

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.

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.

MESP1 loss­of­function mutation contributes to double outlet right ventricle.

Congenital heart disease (CHD) is the most common form of birth defect in humans, and remains a leading non­infectious cause of infant mortality worldwide. An increasing number of studies have demonstrated that genetic defects serve a pivotal role in the pathogenesis of CHD, and mutations in >60 genes have been causally associated with CHD. CHD is a heterogeneous disease and the genetic basis of CHD in the majority of patients remains poorly understood. In the present study, the coding exons and flanking introns of the mesoderm posterior 1 (MESP1) gene, which encodes a basic helix­loop­helix transcription factor required for normal cardiovascular development, were sequenced in 178 unrelated patients with CHD. The available relatives of the index patient carrying an identified mutation and 200 unrelated, ethnically­matched healthy individuals, who were used as controls, were genotyped for MESP1. The functional characteristics of the MESP1 mutation were determined using a dual­luciferase reporter assay system. As a result, a novel de novo heterozygous MESP1 mutation, p.Q118X, was identified in an index patient with double outlet right ventricle (DORV) and a ventricular septal defect. The nonsense mutation was absent in the 400 reference chromosomes and the altered amino acid was completely conserved evolutionarily across species. Functional assays indicated that the mutant MESP1 protein had no transcriptional activity when compared with its wild­type counterpart. The present study firstly provided experimental evidence supporting the concept that a MESP1 loss­of­function mutation may contribute to the development of DORV in humans, which presents a significant insight into the molecular pathogenesis of CHD. The results highlight the potential implications for the genetic counseling and personalized treatment of patients with 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.

Prevalence and spectrum of NKX2.5 mutations in patients with congenital atrial septal defect and atrioventricular block.

Congenital atrial septal defect (ASD) and progressive atriventricular block (AVB) are the two most common phenotypes linked to NK2 homeobox 5 (NKX2.5) mutations in animals and humans. However, the prevalence and spectrum of NKX2.5 mutation in patients with ASD and AVB remain to be elucidated. In the present study, the coding exons and flanking introns of the NKX2.5 gene, which encodes a homeobox­containing transcription factor essential for development of the heart, were sequenced in a cohort of 62 unrelated patients with ASD and AVB, and subsequently in a mutation carrier's available family members. As controls, 300 unrelated, ethnically­matched healthy individuals were recruited, who were also genotyped for NKX2.5. The functional consequence of the mutant NKX2.5 was evaluated in contrast to its wild­type counterpart using a dual­luciferase reporter assay system. As a result, a novel heterozygous NKX2.5 mutation, p.Q181X, was identified in an index patient with ASD and AVB, with a prevalence of ~1.61%. Genetic analysis of the proband's pedigree revealed that the mutation co­segregated with ASD and AVB with complete penetrance. The nonsense mutation, which eliminated partial homeobox and the carboxyl terminus, was absent in the 600 control chromosomes. Functional evaluation showed that the NKX2.5 mutant had no transcriptional activity. Furthermore, the mutation disrupted the synergistic activation between NKX2.5 and GATA binding protein 4, another cardiac core transcription factor associated with ASD. The results of the present study expand the spectrum of NKX2.5 mutations linked to ASD and AVB, and indicated that NKX2.5 loss­of­function mutations are an uncommon cause of ASD and AVB in humans.

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.

HAND1 loss-of-function mutation contributes to congenital double outlet right ventricle.

Congenital heart defects (CHDs), a wide variety of developmental abnormalities in the structures of the heart and the great thoracic blood vessels, are the most common form of birth defect in humans worldwide. CHDs are accountable for substantial morbidity and are still the leading cause of birth defect­related deaths. Recent studies have demonstrated the pivotal roles of genetic defects in the pathogenesis of CHDs, and a great number of genetic mutations have been associated with CHDs. Nevertheless, CHDs are a genetically heterogeneous disorder and the genetic basis underlying CHDs in an overwhelming majority of cases remains unclear. In the present study, the coding exons and flanking introns of the heart and neural crest derivatives expressed transcript 1 (HAND1) gene, which encodes a basic helix­loop­helix transcription factor crucial for cardiovascular development, were sequenced in 158 unrelated patients with CHDs, and a de novo heterozygous mutation, p.K132X, was identified in a patient with double outlet right ventricle (DORV), as well as ventricular septal defect. The nonsense mutation, which was predicted to produce a truncated HAND1 protein lacking 84 carboxyl­terminal amino acids, was absent in 600 control chromosomes. Functional analyses revealed that the HAND1 K132X mutant had no transcriptional activity. Furthermore, the mutation disrupted the synergistic activation between HAND1 and GATA binding protein 4 (GATA4), another cardiac core transcription factor causally linked to CHDs. To the best of our knowledge, this is the first report on the association of HAND1 loss­of­function mutation with an enhanced susceptibility to DORV in humans. These findings expand the phenotypic spectrum linked to HAND1 mutations, suggesting potential implications for the development of novelo prophylactic and therapeutic strategies for DORV.