RESUMO
Defects in laterality pattern can result in abnormal positioning of the internal organs during the early stages of embryogenesis, as manifested in heterotaxy syndrome and situs inversus, while laterality defects account for 3~7% of all congenital heart defects (CHDs). However, the pathogenic mechanism underlying most laterality defects remains unknown. In this study, we recruited 70 laterality defect patients with CHDs to identify candidate disease genes by exome sequencing. We then evaluated rare, loss-of-function (LOF) variants, identifying candidates by referring to previous literature. We chose TRIP11, DNHD1, CFAP74, and EGR4 as candidates from 776 LOF variants that met the initial screening criteria. After the variants-to-gene mapping, we performed function research on these candidate genes. The expression patterns and functions of these four candidate genes were studied by whole-mount in situ hybridization, gene knockdown, and gene rescue methods in zebrafish models. Among the four genes, trip11, dnhd1, and cfap74 morphant zebrafish displayed abnormalities in both cardiac looping and expression patterns of early signaling molecules, suggesting that these genes play important roles in the establishment of laterality patterns. Furthermore, we performed immunostaining and high-speed cilia video microscopy to investigate Kupffer's vesicle organogenesis and ciliogenesis of morphant zebrafish. Impairments of Kupffer's vesicle organogenesis or ciliogenesis were found in trip11, dnhd1, and cfap74 morphant zebrafish, which revealed the possible pathogenic mechanism of their LOF variants in laterality defects. These results highlight the importance of rare, LOF variants in identifying disease-related genes and identifying new roles for TRIP11, DNHD1, and CFAP74 in left-right patterning. Additionally, these findings are consistent with the complex genetics of laterality defects.
Assuntos
Cardiopatias Congênitas , Síndrome de Heterotaxia , Animais , Peixe-Zebra/genética , Peixe-Zebra/metabolismo , Proteínas de Peixe-Zebra/genética , Proteínas de Peixe-Zebra/metabolismo , Padronização Corporal/genética , Cardiopatias Congênitas/metabolismo , Síndrome de Heterotaxia/genética , Síndrome de Heterotaxia/metabolismo , Cílios/genética , Cílios/metabolismoRESUMO
Conotruncal heart defects (CTD), subtypes of congenital heart disease, result from abnormal cardiac outflow tract development (OFT). FOXC1 and FOXC2 are closely related members of the forkhead transcription factor family and play essential roles in the development of OFT. We confirmed their expression pattern in mouse and human embryos, identifying four variants in FOXC1 and three in FOXC2 by screening these two genes in 605 patients with sporadic CTD. Western blot demonstrated expression levels, while Dual-luciferase reporter assay revealed affected transcriptional abilities for TBX1 enhancer in two FOXC1 variants and three FOXC2 variants. This might result from the altered DNA-binding abilities of mutant proteins. These results indicate that functionally impaired FOXC1 and FOXC2 variants may contribute to the occurrence of CTD.
Assuntos
Fatores de Transcrição Forkhead , Cardiopatias Congênitas , Fatores de Transcrição Forkhead/genética , Fatores de Transcrição Forkhead/metabolismo , Humanos , Cardiopatias Congênitas/genética , Cardiopatias Congênitas/metabolismo , Animais , Camundongos , Proteínas com Domínio T/genética , Proteínas com Domínio T/metabolismoRESUMO
OBJECTIVE: Deleterious genetic variants comprise one cause of cardiac conotruncal defects (CTDs). Genes associated with CTDs are gradually being identified. In the present study, we aimed to explore the profile of genetic variants of CTD-associated genes in Chinese patients with non-syndromic CTDs. METHODS: Thirty-nine CTD-related genes were selected after reviewing published articles in NCBI, HGMD, OMIM, and HPO. In total, 605 patients with non-syndromic CTDs and 300 healthy controls, all of Han ethnicity, were recruited. High-throughput targeted sequencing was used to detect genetic variants in the protein-coding regions of genes. We performed rigorous variant-level filtrations to identify potentially damaging variants (Dvars) using prediction programs including CADD, SIFT, PolyPhen-2, and MutationTaster. RESULT: Dvars were detected in 66.7% (26/39) of the targeted CTD-associated genes. In total, 11.07% (67/605) of patients with non-syndromic CTDs were found to carry one or more Dvars in targeted CTD-associated genes. Dvars in FOXH1, TBX2, NFATC1, FOXC2, and FOXC1 were common in the CTD cohort (1.5% [9/605], 1.2% [7/605], 1.2% [7/605], 1% [6/605], and 0.5% [3/605], respectively). CONCLUSION: Targeted exon sequencing is a cost-effective approach for the genetic diagnosis of CTDs. Our findings contribute to an understanding of the genetic architecture of non-syndromic CTDs.
Assuntos
População do Leste Asiático , Cardiopatias Congênitas , Criança , Humanos , População do Leste Asiático/genética , Etnicidade , Cardiopatias Congênitas/genética , Fatores de TranscriçãoRESUMO
BACKGROUND: Heterotaxy syndrome (HTX) is caused by aberrant left-right patterning early in embryonic development, which results in abnormal positioning and morphology of the thoracic and abdominal organs. Currently, genetic testing discerns the underlying genetic cause in less than 20% of sporadic HTX cases, indicating that genetic pathogenesis remains poorly understood. In this study, we aim to garner a deeper understanding of the genetic factors of this disease by documenting the effect of different matrix metalloproteinase 21 (MMP21) variants on disease occurrence and pathogenesis. METHODS: Eighty-one HTX patients with complex congenital heart defects and 89 healthy children were enrolled, and we investigated the pathogenetic variants related to patients with HTX by exome sequencing. Zebrafish splice-blocking Morpholino oligo-mediated transient suppression assays were performed to confirm the potential pathogenicity of missense variants found in these patients with HTX. RESULTS: Three MMP21 heterozygous non-synonymous variants (c.731G > A (p.G244E), c.829C > T (p.L277F), and c.1459A > G (p.K487E)) were identified in three unrelated Chinese Han patients with HTX and complex congenital heart defects. Sanger sequencing confirmed that all variants were de novo. Cell transfection assay showed that none of the variants affect mRNA and protein expression levels of MMP21. Knockdown expression of mmp21 by splice-blocking Morpholino oligo in zebrafish embryos revealed a heart looping disorder, and mutant human MMP21 mRNA (c.731G > A, c.1459A > G, heterozygous mRNA (wild-type&c.731G > A), as well as heterozygous mRNA (wild-type& c.1459A > G) could not effectively rescue the heart looping defects. A patient with the MMP21 p.G244E variant was identified with other potential HTX-causing missense mutations, whereas the patient with the MMP21 p.K487E variant had no genetic mutations in other causative genes related to HTX. CONCLUSION: Our study highlights the role of the disruptive heterozygous MMP21 variant (p.K487E) in the etiology of HTX with complex cardiac malformations and expands the current mutation spectrum of MMP21 in HTX.
Assuntos
Síndrome de Heterotaxia , Animais , Criança , China , Síndrome de Heterotaxia/genética , Humanos , Morfolinos , RNA Mensageiro , Fatores de Risco , Peixe-Zebra/genéticaRESUMO
Long continuous stretches of homozygosity (LCSH) are associated with risk of recessive disorders. Though LCSH can be detected by SNP microarrays, additional testing is necessary to clarify the clinical significance. This study is to assess the yield of additional exome sequencing (ES) after LCSH detection and inform the likelihood of eventual diagnosis. In 2226 patients referred to SNP microarrays, 35 patients met the criteria of indicative LCSH. These patients were recruited and went through additional ES. The diagnostic yield was analyzed, and the LCSH pattern was compared between eventually diagnosed cases and those undiagnosed. The results showed additional ES attained a diagnostic yield of 31.4% (11/35), but only one-third of the yield (11.4%, 4/35) was relevant to LCSH. In contrast, two-thirds of the diagnostic variants (20%, 7/35) were de novo or dominantly inherited, irrelevant to the original LCSH finding. No particular LCSH pattern, including the chromosomal coverage or LCSH size, was found to associate with the diagnostic outcome. We concluded that additional ES after LCSH detection could reveal diagnostic variants, but it is strongly recommended to consider all possible inheritance mode, as the diagnostic variants may be irrelevant to the original LCSH finding.
Assuntos
Genes Recessivos , Doenças Genéticas Inatas/genética , Doenças Genéticas Inatas/patologia , Homozigoto , Polimorfismo de Nucleotídeo Único , Variações do Número de Cópias de DNA , Humanos , Sequenciamento do ExomaRESUMO
The endothelial-to-mesenchymal transition (EndMT) is a critical process that occurs during the development of the outflow tract (OFT). Malformations of the OFT can lead to the occurrence of conotruncal defect (CTD). SOX7 duplication has been reported in patients with congenital CTD, but its specific role in OFT development remains poorly understood. To decipher this, histological analysis showed that SRY-related HMG-box 7 (SOX7) was regionally expressed in the endocardial endothelial cells and in the mesenchymal cells of the OFT, where EndMT occurs. Experiments, using in vitro collagen gel culture system, revealed that SOX7 was a negative regulator of EndMT that inhibited endocardial cell (EC) migration and resulted in decreased number of mesenchymal cells. Forced expression of SOX7 in endothelial cells blocked further migration and improved the expression of the adhesion protein vascular endothelial (VE)-cadherin (VE-cadherin). Moreover, a VE-cadherin knockdown could partly reverse the SOX7-mediated repression of cell migration. Luciferase and electrophoretic mobility shift assay (EMSA) demonstrated that SOX7 up-regulated VE-cadherin by directly binding to the gene's promoter in endothelial cells. The coding exons and splicing regions of the SOX7 gene were also scanned in the 536 sporadic CTD patients and in 300 unaffected controls, which revealed four heterozygous SOX7 mutations. Luciferase assays revealed that two SOX7 variants weakened the transactivation of the VE-cadherin promoter. In conclusion, SOX7 inhibited EndMT during OFT development by directly up-regulating the endothelial-specific adhesion molecule VE-cadherin. SOX7 mutations can lead to impaired EndMT by regulating VE-cadherin, which may give rise to the molecular mechanisms associated with SOX7 in CTD pathogenesis.
Assuntos
Antígenos CD/metabolismo , Caderinas/metabolismo , Endocárdio/embriologia , Cardiopatias Congênitas/embriologia , Fatores de Transcrição SOXF/metabolismo , Animais , Antígenos CD/genética , Caderinas/genética , Movimento Celular , Embrião de Mamíferos , Endocárdio/citologia , Endotélio/crescimento & desenvolvimento , Transição Epitelial-Mesenquimal/fisiologia , Feminino , Regulação da Expressão Gênica no Desenvolvimento , Células Endoteliais da Veia Umbilical Humana , Humanos , Masculino , Camundongos Endogâmicos C57BL , Regiões Promotoras Genéticas , Ratos , Fatores de Transcrição SOXF/genéticaRESUMO
BACKGROUND: Hypertrophic cardiomyopathy (HCM) is the most common inheritable cardiac disease and is characterised by unexplained ventricular myocardial hypertrophy. HCM is highly heterogeneous and is primarily caused by the mutation of genes encoding sarcomere proteins. As a result of its genetic basis, we investigated the underlying cause of HCM in a Chinese family by whole-exome sequencing. METHODS: Whole-exome sequencing was performed for seven clinically diagnosed HCM family members and the resulting single nucleotide variants associated with cardiac hypertrophy or heart development were analysed by a polymerase chain reaction and Sanger sequencing. RESULTS: A non-frameshift deletion mutation (p.S527del) of Formin Homology 2 Domain Containing 3 (FHOD3) was detected in all of the affected family members and was absent in all unaffected members, with the exception of one young member. Moreover, three single nucleotide variants associated with heart development and morphogenesis were identified in the proband but were absent in the other affected subjects. CONCLUSIONS: This is the first HCM family case of FHOD3 (p.S527del) variation in Asia. Additionally, RNF207 (p.Q268P), CCM2 (p. E233K) and SGCZ (p.Q134X) may be related to the clinical heterogeneity of the family. The present study could enable the provision of genetic counseling for this family and provide a basis for future genetic and functional studies.
Assuntos
Cardiomiopatia Hipertrófica/genética , Exoma , Forminas/genética , Povo Asiático , Cardiomiopatia Hipertrófica/diagnóstico , Feminino , Predisposição Genética para Doença , Variação Genética , Humanos , Masculino , Mutação , Linhagem , Reação em Cadeia da Polimerase , Polimorfismo de Nucleotídeo Único , Deleção de Sequência , Sequenciamento do ExomaRESUMO
Conotruncal heart defects (CTD) are an important subtype of congenital heart disease that occur due to abnormality in the development of the cardiac outflow tract (OFT). FOXH1 is a transcription factor that participates in the morphogenesis of the right ventricle and OFT. In this study, we confirmed the expression of FOXH1 in mouse and human embryos during OFT development. We also scanned the coding exons and splicing regions of the FOXH1 gene in 605 patients with sporadic CTD and 300 unaffected controls, from which we identified seven heterozygous FOXH1 gene mutations. According to bioinformatics analysis results, they were predicted potentially deleterious at conserved amino acid sites. Western blot was used to show that all the variants decreased the expression of FOXH1 protein, while dual-luciferase reporter assay showed that six of them, with an exception of p.P35R, had enhanced abilities to modulate the expression of MEF2C, which interacts with NKX2.5 and is involved in cardiac growth. The electrophoretic mobility shift assays result showed that two mutations altered DNA-binding abilities of mutant FOXH1 proteins. Phenotype heterogeneity was found in patients with the same mutation. These results indicate that FOXH1 mutations lead to disease-causing functional changes that contribute to the occurrence of CTD.
Assuntos
Fatores de Transcrição Forkhead/genética , Cardiopatias Congênitas/genética , Proteína Homeobox Nkx-2.5/genética , Fatores de Transcrição MEF2/genética , Animais , Modelos Animais de Doenças , Feminino , Regulação da Expressão Gênica , Predisposição Genética para Doença , Cardiopatias Congênitas/patologia , Humanos , Lactente , Recém-Nascido , Masculino , Camundongos , Mutação de Sentido Incorreto/genéticaRESUMO
Conotruncal heart defects (CTDs) are a group of cardiac malformations that involve outflow tract anomalies and the arterial pole of the heart. Recent reports have identified mutations in a number of genes associated with CTDs in human and animal models. ZFPM2 plays a role in cardiac development by acting as a transcriptional cofactor that interacts with GATA4. Because ZFPM2 was found to be important for cardiac development in a knockout mouse model, we screened for ZFPM2 mutations in 528 CTD patients. We identified six rare and nonsynonymous ZFPM2 variants, and this was the first time that five of these variants (R698Q, R736L, E1005K, T32A, and I488V) were reported in East Asians. Western blots showed that there was no significant difference in the protein expression of wild-type ZFPM2, ZFPM2R698Q, or ZFPM2R736L. A dual luciferase reporter assay demonstrated that both ZFPM2 mutants R698Q and R736L reduced GATA4-mediated transcription. However, when ZFPM2R698Q was co-transfected with GATA4, BNP promoter activity increased significantly, whereas co-transfection with ZFPM2R736L and GATA4 did not significantly increase BNP promoter activity. This suggests that the R698Q mutation may affect the ability of ZFPM2 to bind GATA4.
Assuntos
Proteínas de Ligação a DNA/genética , Fator de Transcrição GATA4/genética , Predisposição Genética para Doença , Cardiopatias Congênitas/genética , Fatores de Transcrição/genética , Animais , Análise Mutacional de DNA , Proteínas de Ligação a DNA/metabolismo , Feminino , Fator de Transcrição GATA4/metabolismo , Cardiopatias Congênitas/metabolismo , Cardiopatias Congênitas/patologia , Humanos , Masculino , Camundongos , Mutação de Sentido Incorreto , Ligação Proteica , Fatores de Transcrição/metabolismoRESUMO
BACKGROUND: Conotruncal heart defect (CTD) is a complex congenital heart disease with a complex and poorly understood etiology. The transcriptional corepressor RIPPLY3 plays a pivotal role in heart development as a negative regulator of the key cardiac transcription factor TBX1. A previous study showed that RIPPLY3 contribute to cardiac outflow tract development in mice, however, the relationship between RIPPLY3 and human cardiac malformation has not been reported. METHODS: 615 unrelated CTD Chinese Han patients were enrolled, we excluded the 22q11.2 deletion/duplication using a modified multiplex ligation-dependent probe amplification method-CNVplex®, and investigated the variants of RIPPLY3 in 577 patients without the 22q11.2 deletion/duplication by target sequencing. Functional assays were performed to testify the potential pathogenicity of nonsynonymous variants found in these CTD patients. RESULTS: Four rare heterozygous nonsynonymous variants (p.P30L, p.T52S, p.D113N and p.V179D) were identified in four CTD patients, the variant NM_018962.2:c.155C>G (p.T52S) is referred as rs745539198, and the variant NM_018962.2:c.337G>A (p.D113N) is referred as rs747419773. However, variants p.P30L and p.V179D were not found in multiple online human gene variation databases. Western blot analysis and immunofluorescence showed that there were no significant difference between wild type RIPPLY3 and these four variants. Luciferase assays revealed that the p.T52S variant altered the inhibition of TBX1 transcriptional activity in vitro, and co-immunoprecipitation assays showed that the p.T52S variant reduced the physical interaction of RIPPLY3 with TBX1. In addition to the results from pathogenicity prediction tools and evolutionary protein conservation, the p.T52S variant was thought to be a potentially deleterious variant. CONCLUSION: Our results provide evidence that deleterious variants in RIPPLY3 are potential molecular mechanisms involved in the pathogenesis of human CTD.
Assuntos
Anormalidades Múltiplas/genética , Povo Asiático/genética , Duplicação Cromossômica/genética , Síndrome de DiGeorge/genética , Etnicidade/genética , Predisposição Genética para Doença , Cardiopatias Congênitas/genética , Mutação com Perda de Função/genética , Proteínas Repressoras/genética , Fatores de Transcrição/genética , Sequência de Aminoácidos , Cromossomos Humanos Par 22/genética , Estudos de Coortes , Variações do Número de Cópias de DNA/genética , Células HEK293 , Humanos , Mutação de Sentido Incorreto/genética , Ligação Proteica , Proteínas Repressoras/química , Fatores de Risco , Proteínas com Domínio T/genética , Transcrição GênicaRESUMO
Conotruncal heart defects (CTDs) represent a group of severe and complicated congenital cardiovascular malformations and require opportune clinical interventions once diagnosed. Occurrence of CTD is related to the functional abnormality of the second heart field (SHF), and variants of genes which regulate the development of the second heart field have been recognized as the main genetic factors leading to CTDs. Previous studies indicated that transcriptional complex SIX1/EYA1 may contribute to SHF development, and SIX1/EYA1 knockout mice exhibited a series of conotruncal malformations. Here, we recruited and sequenced 600 Chinese conotruncal heart defect patients and 300 controls. We screened out one novel SIX1 mutation (SIX1-K134R) and four EYA1 rare mutations (EYA1-A227T, EYA1-R296H, EYA1-Q397R, EYA1-G426S), all variants were present only in the case cohort, and the mutated sites were highly conserved. We then analyzed mutations by software including Sift, PolyPhen-2, PROVEAN, Mutation Taster, HOPE, and SWISS-PdbViewer. The results showed that the mutations had varying degrees of pathogenic risk, protein properties, spatial conformations, and domain functions which might be altered or influenced. Through biological and in silico analyses, our study suggests an association between SIX1/EYA1 mutations and cardiovascular malformations, SIX1/EYA1 mutations might be partially responsible for CTDs.
Assuntos
Cardiopatias Congênitas/genética , Proteínas de Homeodomínio/genética , Peptídeos e Proteínas de Sinalização Intracelular/genética , Proteínas Nucleares/genética , Proteínas Tirosina Fosfatases/genética , Povo Asiático/genética , Simulação por Computador , Análise Mutacional de DNA , Predisposição Genética para Doença , Humanos , Masculino , MutaçãoRESUMO
Anomalous pulmonary venous connection (APVC) is an uncommon congenital anomaly in which pulmonary venous blood flows directly into the right side of the heart or into the systemic veins. To identify whether there is any association between 22q11 CNVs and APVC, we analyzed the clinical data of 86 APVC patients and then studied the CNVs of 22q11 in 86 sporadic APVC patients by multiplex ligation-dependent probe amplification. The results showed that two patients carried the CNVs of 22q11, one patient had the deletion of 22q11 and the other had the duplication of 22q11. The incidence was significantly higher than that in the normal population (P < 0.01) that suggests a possible etiologic association between the duplication or deletion of 22q11 and the APVC in our patients.
Assuntos
Deleção Cromossômica , Duplicação Cromossômica , Cromossomos Humanos Par 22/genética , Síndrome de Cimitarra/genética , Criança , Variações do Número de Cópias de DNA , Feminino , Humanos , Masculino , Reação em Cadeia da Polimerase Multiplex , Síndrome de Cimitarra/etiologiaRESUMO
Congenital heart disease (CHD) is one of the most common birth defects. More than 200 susceptibility loci have been identified for CHDs, yet a large part of the genetic risk factors remain unexplained. Monozygotic (MZ) twins are thought to be completely genetically identical; however, discordant phenotypes have been found in MZ twins. Recent studies have demonstrated genetic differences between MZ twins. We aimed to test whether copy number variants (CNVs) and/or genetic mutation differences play a role in the etiology of CHDs by using single nucleotide polymorphism (SNP) genotyping arrays and whole exome sequencing of twin pairs discordant for CHDs. Our goal was to identify mutations present only in the affected twins, which could identify novel candidates for CHD susceptibility loci. We present a comprehensive analysis for the CNVs and genetic mutation results of the selected individuals but detected no consistent differences within the twin pairs. Our study confirms that chromosomal structure or genetic mutation differences do not seem to play a role in the MZ twins discordant for CHD.
Assuntos
Variações do Número de Cópias de DNA/genética , Doenças em Gêmeos/genética , Cardiopatias Congênitas/genética , Gêmeos Monozigóticos/genética , Adulto , China/epidemiologia , Doenças em Gêmeos/patologia , Exoma/genética , Feminino , Genótipo , Cardiopatias Congênitas/patologia , Humanos , Recém-Nascido , Masculino , Pessoa de Meia-Idade , Mutação , Sequenciamento do ExomaRESUMO
BACKGROUND/AIMS: Cardiac myocytes constitute the first differentiated cell type during mammalian heart formation with the ability to beat spontaneously and rhythmically. Hyperglycemia is a primary risk factor for cardiovascular disease in pre-gestational diabetes mellitus (PGDM). However, the impact that hyperglycemia has on cardiac progenitors or on precursors differentiation remains poorly understood. The aim of the present study is to investigate whether hyperglycemia affects cardiomyogenesis of embryocarcinoma cells. METHODS: P19CL6 cells differentiation induced by 1% DMSO was evaluated under either normal glucose (5.6 mmol/L) or high level of glucose concentrations (20 mmol/L or 40 mmol/L). To investigate the effect of long-term high level of glucose on cardiomyocytes differentiation, sarcomeric α-actinin, peroxisome proliferator-activated receptor coactivator-1 (PGC-1α), transcription factor GATA4 and Nkx2.5 were assessed by qRT-PCR analysis, western blot and immunofluorescence. RESULTS: We observed that long-term high level of glucose markedly reduced P19CL6 cells differentiation into cardiomyocytes. The change in PGC-1α expression was consistent with changes in cardiac muscle myosin expression after exposure to 20 mmol/L or 40 mmol/L of glucose. On the other hand, the high level of glucose concentration profoundly decreased both GATA4 and Nkx2-5 expressions from day 6 to day 12 after differentiation, which was induced by 1% DMSO. CONCLUSION: Our results elucidate that the effect resulting from the long-term exposure of cardiac progenitors to high level of glucose is associated with decreased expression of GATA4 and Nkx2.5, providing a novel mechanism by which high glucose is able to affect cell differentiation.
Assuntos
Diferenciação Celular/efeitos dos fármacos , Glucose/farmacologia , Actinina/genética , Actinina/metabolismo , Animais , Western Blotting , Linhagem Celular Tumoral , Dimetil Sulfóxido/farmacologia , Fator de Transcrição GATA4/genética , Fator de Transcrição GATA4/metabolismo , Proteína Homeobox Nkx-2.5/genética , Proteína Homeobox Nkx-2.5/metabolismo , Camundongos , Microscopia de Fluorescência , Miócitos Cardíacos/citologia , Miócitos Cardíacos/metabolismo , Coativador 1-alfa do Receptor gama Ativado por Proliferador de Peroxissomo/genética , Coativador 1-alfa do Receptor gama Ativado por Proliferador de Peroxissomo/metabolismo , Reação em Cadeia da Polimerase em Tempo RealRESUMO
BACKGROUND: Cornelia de Lange Syndrome (CdLS) is a rare but severe clinically heterogeneous developmental disorder characterized by facial dysmorphia, growth and cognitive retardation, and abnormalities of limb development. OBJECTIVES: To determine the pathogenesis of a patient with CdLS. METHODS: We studied a patient with CdLS by whole exome sequencing, karyotyping and Agilent CGH Array. The results were confirmed by quantitative real-time PCR analysis of the patient and her parents. Further comparison of our patient and cases with partially overlapping deletions retrieved from the literature and databases was undertaken. RESULTS: Whole exome sequencing had excluded the mutation of cohesion genes such as NIPBL,SMC1A and SMC3. The result of karyotyping showed a deletion of chromosome 9q31.1-q32 and the result of Agilent CGH Array further displayed a 12.01-Mb region of deletion at chromosome bands 9q31.1-q32. Reported cases with the deletion of 9q31.1-q32 share similar features with our CdLS patient. One of the genes in the deleted region, SMC2, belongs to the Structural Maintenance of Chromosomes (SMC) family and regulates gene expression and DNA repair. CONCLUSIONS: Patients carrying the deletion of 9q31.1-q32 showed similar phenotypes with CdLS.
Assuntos
Cromossomos Humanos Par 9 , Síndrome de Cornélia de Lange/genética , Hibridização Genômica Comparativa , Síndrome de Cornélia de Lange/patologia , Ecocardiografia , Feminino , Sequenciamento de Nucleotídeos em Larga Escala , Humanos , Lactente , Cariotipagem , Fenótipo , Análise de Sequência de DNARESUMO
BACKGROUND: TBX1 and CRKL haploinsufficiency is thought to cause the cardiac phenotype of the 22q11.2 deletion syndrome. However, few unequivocal mutations of TBX1 and CRKL have been discovered in isolated conotrucal heart defects (CTDs) patients. The aim of the study was to screen the mutation of TBX1 and CRKL in isolated CTDs Chinese patients without 22q11.2 deletion and identify the pathomechanism of the missense mutations. METHODS: We enrolled 199 non-22q11.2 deletion patients with CTDs and 139 unrelated healthy controls. Gene sequencing were performed for all of them. The functional data of mutations were obtained by in vitro transfection and luciferase experiments and computer modelling. RESULTS: Screening of the TBX1 coding sequence identified a de novo missense mutation (c.385G â A; p.E129K) and a known polymorphism (c.928G â A; p.G310S). In vitro experiments demonstrate that the TBX1E129K variant almost lost transactivation activity. The TBX1G310S variant seems to affect the interaction of TBX1 with other factors. Computer molecular dynamics simulations showed the de novo missense mutation is likely to affect TBX1-DNA interaction. No mutation of CRKL gene was found. CONCLUSIONS: These observations suggest that the TBX1 loss-of-function mutation may be involved in the pathogenesis of isolated CTDs. This is the first human missense mutation showing that TBX1 is a candidate causing isolated CTDs in Chinese patients without 22q11.2 deletion.
Assuntos
Proteínas Adaptadoras de Transdução de Sinal/genética , Síndrome de DiGeorge/genética , Cardiopatias Congênitas/genética , Proteínas Nucleares/genética , Proteínas com Domínio T/genética , Proteínas com Domínio T/metabolismo , Animais , Povo Asiático/genética , Células COS , Estudos de Casos e Controles , Chlorocebus aethiops , DNA/química , DNA/metabolismo , Síndrome de DiGeorge/patologia , Éxons , Feminino , Células HEK293 , Cardiopatias Congênitas/patologia , Humanos , Masculino , Simulação de Dinâmica Molecular , Mutação de Sentido Incorreto , Filogenia , Conformação Proteica , Estrutura Secundária de Proteína , Análise de Sequência de DNA , Proteínas com Domínio T/químicaRESUMO
Most viruses and transposons serve as effective carriers for the introduction of foreign DNA up to 11 kb into vertebrate genomes. However, their activity markedly diminishes with payloads exceeding 11 kb. Expanding the payload capacity of transposons could facilitate more sophisticated cargo designs, improving the regulation of expression and minimizing mutagenic risks associated with molecular therapeutics, metabolic engineering, and transgenic animal production. In this study, we improved the Tol2 transposon by increasing protein expression levels using a translational enhancer ( QBI SP163, ST) and enhanced the nuclear targeting ability using the nuclear localization protein H2B (SHT). The modified Tol2 and ST transposon efficiently integrated large DNA cargos into human cell cultures (H1299), comparable to the well-established super PiggyBac system. Furthermore, mRNA from ST and SHT showed a significant increase in transgene delivery efficiency of large DNA payloads (8 kb, 14 kb, and 24 kb) into zebrafish ( Danio rerio). This study presents a modified Tol2 transposon as an enhanced nonviral vector for the delivery of large DNA payloads in transgenic applications.
Assuntos
Elementos de DNA Transponíveis , Transgenes , Peixe-Zebra , Animais , Peixe-Zebra/genética , Elementos de DNA Transponíveis/genética , Humanos , Animais Geneticamente Modificados , Técnicas de Transferência de GenesRESUMO
Laterality is a crucial physiological process intricately linked to the cilium-centrosome complex during embryo development. Defects in the process can result in severe organ mispositioning. Coiled-coil domain containing 141 (CCDC141) has been previously known as a centrosome-related gene, but its role in left-right (LR) asymmetry has not been characterized. In this study, we utilize the zebrafish model and human exome analysis to elucidate the function of ccdc141 in laterality defects. The knockdown of ccdc141 in zebrafish disrupts early LR signaling pathways, cilia function, and Kupffer's vesicle (KV) formation. Unlike ccdc141-knockdown embryos exhibiting aberrant LR patterns, ccdc141-null mutants show no apparent abnormality, suggesting a genetic compensation response effect. In parallel, we observe a marked reduction in α-tubulin acetylation levels in the ccdc141 crispants. The treatment with histone deacetylase (HDAC) inhibitors, particularly the HDAC6 inhibitor, rescues the ccdc141 crispant phenotypes. Furthermore, exome analysis of 70 patients with laterality defects reveals an increased burden of CCDC141 mutations, with in-vivo studies verifying the pathogenicity of the patient mutation CCDC141-R123G. Our findings highlight the critical role of CCDC141 in ciliogenesis and demonstrate that CCDC141 mutations lead to abnormal LR patterns. Thus, we identify CCDC141 as a causative gene for laterality defects.
RESUMO
AIM: Hypoxia, a characteristic of almost all types of solid tumors, has been associated with poor outcome in a number of human malignancies. The aim of this study was to investigate the molecular mechanisms involved in hypoxia-induced activation of the human survivin gene promoter in cervical HeLa cells. MATERIAL AND METHODS: Immunohistochemical staining was used to detect the expression of HIF-1α and survivin in cervical cancer samples and normal cervical samples. Under normoxic and hypoxic conditions, the expression of hypoxia inducible factor (HIF)-1α and survivin in cervical cancer HeLa cells was detected by quantitative reverse transcription polymerase chain reaction and Western blotting. Luciferase reporter assays was used to investigate the molecular mechanisms in hypoxia-induced survivin activation. We also studied the effect of HIF-1α overexpression on the expression of survivin in cervical cancer HeLa cells. RESULTS: Significant HIF-1α and survivin overexpression is associated with cervical cancer, and HIF-1α protein expression is strongly correlated with survivin protein expression. In cervical cancer cell line (HeLa), hypoxia upregulated both HIF-1α and survivin expression. Moreover, luciferase reporter assays using survivin core promoter demonstrated that survivin transcription was activated under hypoxia conditions and was associated with HIF-1α overexpression. The transcriptional activation of reporter genes in response to hypoxia is independent of potential HIF-1α-responsive element, located between -86 and -82 regions. HIF-1α overexpression significantly activated survivin expression. CONCLUSION: Our results demonstrate that survivin expression is upregulated following the induction of HIF-1α by hypoxia resulting from tumor formation, possibly leading to tumor progression. These findings have potential implication in developing novel cancer therapy targeting HIF-1.
Assuntos
Carcinoma/metabolismo , Colo do Útero/metabolismo , Subunidade alfa do Fator 1 Induzível por Hipóxia/metabolismo , Proteínas Inibidoras de Apoptose/metabolismo , Proteínas de Neoplasias/metabolismo , Regulação para Cima , Neoplasias do Colo do Útero/metabolismo , Idoso , Carcinoma/patologia , Hipóxia Celular , Linhagem Celular Tumoral , Colo do Útero/patologia , Feminino , Humanos , Proteínas Inibidoras de Apoptose/biossíntese , Proteínas Inibidoras de Apoptose/genética , Pessoa de Meia-Idade , Mutação , Proteínas de Neoplasias/biossíntese , Proteínas de Neoplasias/genética , Estadiamento de Neoplasias , Regiões Promotoras Genéticas , Survivina , Neoplasias do Colo do Útero/patologiaRESUMO
It is well known that activation of the phagocyte NADPH oxidase requires the association of cytosolic proteins (p67-phox, p47-phox, p40-phox, and Rac) with the membrane cytochrome b(558), leading to its conformation change. Recently, the phagocyte NADPH oxidase complex was isolated in a constitutively active form. In this complex, 6-phosphogluconate dehydrogenase (6PGDH), an enzyme involved in the production of intracellular NADPH, was identified. This protein was absent from the oxidase complex isolated from B lymphocytes, suggesting a specific interaction with the neutrophil NADPH oxidase. To clarify the implication of 6PGDH in the NADPH oxidase activity, a siRNA approach was conducted in neutrophil-like PLB985 cells. NADPH oxidase activity of siRNA-transfected cells was shown to be decreased. Similar results were obtained in vitro, after reconstitution of oxidase activity with subcellular fractions isolated from siRNA-transfected cells. Interestingly, the Michaelis constant (K(m)) of Nox2 for NADPH increases in 6PGDH-depleted cells. Moreover, 6PGDH coimmunoprecipitated with oxidase cytosolic factors from cytosol of stimulated cells. Data suggested that the affinity of Nox2 for NADPH is increased in the presence of 6PGDH on cell stimulation. The present work proposes a new way of NADPH oxidase activity regulation by modulating Nox2 affinity for NADPH.