Variants in FOXC1 and FOXC2 identified in patients with conotruncal heart defects.

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.

SOX7 suppresses endothelial-to-mesenchymal transitions by enhancing VE-cadherin expression during outflow tract development.

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.

Identification of FOXH1 mutations in patients with sporadic conotruncal heart defect.

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.

In Silico Analyses Reveal the Relationship Between SIX1/EYA1 Mutations and Conotruncal Heart Defects.

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.

Mutation Screening of Gata4 Gene in CTD Patients Within Chinese Han Population.

Conotruncal heart defect is a complex form of congenital heart disease and usually has a poor prognosis. Although previous studies have identified several missense variants in GATA4 gene that may cause CTD, it remains unclear whether they are involved in CTD pathogenesis because the study population was limited. The aim of the study was to investigate the mutations of GATA4 gene in isolated CTD Chinese Han patients and identify the pathomechanism of the missense mutations. In this report, the coding exons and exon-intron boundaries of the GATA4 gene were sequenced in 600 CTD patients and 300 controls. Functional significance of the novel GATA4 gene mutation (p.A167D) was analyzed using PolyPhen 2 and SIFT. And, the functional characteristics of the mutant GATA4 gene were assayed in contrast to its wild-type counterpart using a luciferase reporter assay system as well as Western blot. Eight heterozygous nonsynonymous variants (V380M, G64E, A167D, V267M, S377G, P163S, P407Q, A66T) were found in 22 patients, of which one (A167D) was reported here for the first time and five (G64E, A167D, S377G, P163S, A66T) were only found in CTD patients when compared with 300 controls. The PolyPhen 2 and SIFT programs predicted that the A167D substitution was expected to influence protein function. Subsequent functional analyses revealed that the transcriptional activity and Western blot of A167D mutant GATA4 protein were not altered. These variants may be involved in other mechanisms underlying CTD or may be unrelated to CTD occurrence.

Image Intrinsic-Based Unsupervised Monocular Depth Estimation in Endoscopy.

Unsupervised monocular depth estimation plays a vital role for endoscopy-based minimally invasive surgery (MIS). However, it remains challenging due to the distinctive imaging characteristics of endoscopy which disrupt the assumption of photometric consistency, a foundation relied upon by conventional methods. Distinct from recent approaches taking image pre-processing strategy, this paper introduces a pioneering solution through intrinsic image decomposition (IID) theory. Specifically, we propose a novel end-to-end intrinsic-based unsupervised monocular depth learning framework that is comprised of an image intrinsic decomposition module and a synthesis reconstruction module. This framework seamlessly integrates IID with unsupervised monocular depth estimation, and dedicated losses are meticulously designed to offer robust supervision for network training based on this novel integration. Noteworthy, we rely on the favorable property of the resulting albedo map of IID to circumvent the challenging images characteristics instead of pre-processing the input frames. The proposed method is extensively validated on SCARED and Hamlyn datasets, and better results are obtained than state-of-the-art techniques. Beside, its generalization ability and the effectiveness of the proposed components are also validated. This innovative method has the potential to elevate the quality of 3D reconstruction in monocular endoscopy, thereby enhancing the accuracy and robustness of augmented reality navigation technology in MIS. Our code will be available at: https://github.com/bobo909/IID-SfmLearner.

RT-SRTS: Angle-agnostic real-time simultaneous 3D reconstruction and tumor segmentation from single X-ray projection.

Radiotherapy is one of the primary treatment methods for tumors, but the organ movement caused by respiration limits its accuracy. Recently, 3D imaging from a single X-ray projection has received extensive attention as a promising approach to address this issue. However, current methods can only reconstruct 3D images without directly locating the tumor and are only validated for fixed-angle imaging, which fails to fully meet the requirements of motion control in radiotherapy. In this study, a novel imaging method RT-SRTS is proposed which integrates 3D imaging and tumor segmentation into one network based on multi-task learning (MTL) and achieves real-time simultaneous 3D reconstruction and tumor segmentation from a single X-ray projection at any angle. Furthermore, the attention enhanced calibrator (AEC) and uncertain-region elaboration (URE) modules have been proposed to aid feature extraction and improve segmentation accuracy. The proposed method was evaluated on fifteen patient cases and compared with three state-of-the-art methods. It not only delivers superior 3D reconstruction but also demonstrates commendable tumor segmentation results. Simultaneous reconstruction and segmentation can be completed in approximately 70 ms, significantly faster than the required time threshold for real-time tumor tracking. The efficacies of both AEC and URE have also been validated in ablation studies. The code of work is available at https://github.com/ZywooSimple/RT-SRTS.

[Horizontal Variation Characteristics of Alpine Grassland Soil Function and Vertical Changes Along Soil Genetic Horizons in the Three-River Headwaters Region].

The Three-River Headwaters region is a hotspot for studying the response of soil function to climate change. To study the horizontal variation characteristics of alpine grassland soil function and vertical changes along soil genetic horizons, soil functional indicators (including respiration, nitrogen conversion rate, and enzymatic activity) of different genetic horizons in alpine grassland soil profiles and their correlations with environmental factors were analyzed. The results showed that there were no significant differences in soil functional characteristics between alpine meadows and steppes, and topsoil had higher respiration rates, nitrogen conversion rates, and enzymatic activities than those of subsoil. Total nitrogen was a key driver of soil functional characteristics in different genetic horizons, explaining 18.3%, 21.4%, and 27.5% of the horizontal variation in functional characteristics, respectively. Climate and vegetation factors mainly affected soil function indirectly by changing soil physicochemical properties in topsoil, but atmospheric nitrogen deposition still affected soil function in subsoil. These results indicate the significant nitrogen limitation of alpine grassland soil in the Three-River Headwaters region, and the findings provide a new insight into the maintenance of soil functional diversity and the response to climate change in the context of global climate change.

Environmental selection dominates over dispersal limitation in shaping bacterial biogeographical patterns across different soil horizons of the Qinghai-Tibet Plateau.

Soil microbial biogeographical patterns have been widely explored from horizontal to vertical scales. However, studies of microbial vertical distributions were still limited (e.g., how soil genetic horizons influence microbial distributions). To shed light on this question, we investigated soil bacterial communities across three soil horizons (topsoil: horizon A; midsoil: horizon B; subsoil: horizon C) of 60 soil profiles along a 3500 km transect in the Qinghai-Tibet Plateau. We found that bacterial diversity was highest in the topsoil and lowest in the subsoil, and community composition significantly differed across soil horizons. The network complexity decreased from topsoil to subsoil. There were significant geographical/environmental distance-decay relationships (DDR) in three soil horizons, with a lower slope from topsoil to subsoil due to the decreased environmental heterogeneity. Variation partitioning analysis (VPA) showed that bacterial community variations were explained more by environmental than spatial factors. Although environmental selection processes played a dominant role, null model analysis revealed that deterministic processes (mainly variable selection) decreased with deeper soil horizons, while stochastic processes (mainly dispersal limitation) increased from topsoil to subsoil. These results suggested that microbial biogeographical patterns and community assembly processes were soil horizon dependent. Our study provides new insights into the microbial vertical distributions in large-scale alpine regions and highlights the vital role of soil genetic horizons in affecting microbial community assembly, which has implications for understanding the pedogenetic process and microbial responses to extreme environment under climate change.

Association between gene polymorphism of GRK5 and breast cancer risk in Chinese population.

PURPOSE: Potential influences of GRK5 polymorphism on cancer risks have been reported. This study aimed to explore the distribution of GRK5 genotypes and alleles in Chinese breast cancer (BCa) patients, and to analyze the association between GRK5 and BCa risk. METHODS: Blood samples were collected from 412 BCa patients and 533 healthy individuals for isolating genomic DNA. GRK5 polymorphisms of Gln41Leu A > T and Arg304His G > A, and their alleles were detected using PCR-RFLP. Their influences on BCa susceptibility and pathological indexes were analyzed using Logistic regression model. RESULTS: No significant differences in age, body mass index (BMI) and smoking status were found between BCa patients and healthy persons, while significant differences were detected in drinking status, family history of cancer, hypertension and diabetes. GRK5Gln41Leu A > T and its allele frequency distribution were correlated to BCa susceptibility, while GRK5 Arg304His G > A was not. Higher risks of GRK5 Gln41Leu A > T and Arg304His G > A indicated a higher susceptibility to BCa. Compared with people carrying 0-1 risk allele, those carrying 2-4 risk alleles of GRK5 Gln41Leu A > T and Arg304His G > A of had a higher susceptibility to BCa, manifesting as worse tumor staging and grading, and higher rates of estrogen receptor (ER) (-), progesterone receptor (PR) (-) and HER2 (-). CONCLUSIONS: Gln41Leu A > T and Arg304His G > A fusion gene polymorphisms of GRK5 are vital genetic susceptibility genes to BCa. Our findings require to be validated in a multicenter study with a high-quality large sample size.

Genetic and functional analyses detect one pathological NFATC1 mutation in a Chinese tricuspid atresia family.

BACKGROUND: Cardiac valvulogenesis is a highly conserved process among vertebrates and cause unidirectional flow of blood in the heart. It was precisely regulated by signal pathways such as VEGF, NOTCH, and WNT and transcriptional factors such as TWIST1, TBX20, NFATC1, and SOX9. Tricuspid atresia refers to morphological deficiency of the valve and confined right atrioventricular traffic due to tricuspid maldevelopment, and is one of the most common types of congenital valve defects. METHODS: We recruited a healthy couple with two fetuses aborted due to tricuspid atresia and identified related gene mutations using whole-exome sequencing. We then discussed the pathogenic significance of this mutation by bioinformatic and functional analyses. RESULTS: PROVEAN, PolyPhen, MutationTaster, and HOPE indicated the mutation could change the protein function and cause disease; Western blotting showed the expression of NFATC1 c.964G>A mutation was lower than the wild type. What's more, dual-luciferase reporter assay showed the transcriptional activity of NFATC1 was impact by mutation and the expression of downstream DEGS1 was influenced. CONCLUSION: Taken together, the c.964G>A mutation might be pathological and related to the occurrence of disease. Our research tended to deepen the understanding of etiology of tricuspid atresia and gene function of NFATC1, and provide some references or suggestions for genetic diagnosis of tricuspid atresia.

Genetic Analyses Identified a SALL4 Gene Mutation Associated with Holt-Oram Syndrome.

Holt-Oram syndrome (HOS) is an autosomal dominant disorder, which is characterized by deformities of upper limbs and congenital heart defects. Alterations of TBX5 gene have been identified to be the leading cause of HOS, while some cases could not be explained by TBX5 mutations. In our study, we preliminarily diagnosed a newborn baby, who had Tetralogy of Fallot, thumb agenesis, facial dysplasia, and right ear canal malformation, as HOS. Chromosome microarray analyses showed no pathological deletions or replications of chromosome segments; whole exome sequencing screened out six candidate genes that were involved in cardiac diseases or syndromes among which SALL4 has been reported as HOS related gene. We evaluated the pathogenicity of SALL4 mutant sites by series of software. The results indicated that SALL4-M143V may be a polymorphism site, and SALL4-R418C could cause disease. HOPE and SWISS PDB viewer showed that SALL4-R418C leads to changes in amino acid properties, loss of protein hydrogen bond, and functional impact of SALL4 zinc finger domain. These results further confirmed the pathogenic significance of SALL4-R418C mutant. When genetic analyses coupled with bioinformatic analyses, we identified a SALL4 gene rare mutation which might contribute to a newborn with HOS. Although some doubts need to be further discussed and explored, our study deepened the understanding of phenotype difference among syndromes and role of SALL4 mutations in disease occurrence.

CITED2 Mutations in Conserved Regions Contribute to Conotruncal Heart Defects in Chinese Children.

Conotruncal heart defects (CTDs) are severe malformations of outflow tract with heterogeneous morphology. Several missense variants of CITED2 have been identified to cause CTDs in recent researches. In this study, we screened the coding regions of CITED2 in 605 Chinese children with CTDs and found two possible pathogenic mutant sites: p.Q117L and p.T257A, both located in the conserved regions of CITED2. Then, we investigated the biological and functional alterations of them. Western blotting showed low level of protein expression of mutant Q117 and T257A compared with wild-type CITED2. Dual-luciferase reporter assay demonstrated that mutant Q117 and T257A decreased the ability of CITED2 to modulate the expression of paired-like homeodomain transcription factor 2 gamma (PITX2C), which are closely related to cardiac growth and left-right patterning. Meanwhile, T257A also exhibited impaired ability to mediate vascular endothelial growth factor expression, another gene closely associated with the normal development of cardiovascular system. Three-dimensional molecular conformation showed reduced hydrogen bond between Asp254 and mutant Thr257, indicating the weakened stability and binding ability of CITED2. All these results suggest that CITED2 mutations in conserved regions lead to disease-causing biological and functional changes and may contribute to the occurrence of CTDs.