Identification of novel M2 macrophage-related molecule ATP6V1E1 and its biological role in hepatocellular carcinoma based on machine learning algorithms.

Hepatocellular carcinoma (HCC) remains the most prevalent form of primary liver cancer, characterized by late detection and suboptimal response to current therapies. The tumour microenvironment, especially the role of M2 macrophages, is pivotal in the progression and prognosis of HCC. We applied the machine learning algorithm-CIBERSORT, to quantify cellular compositions within the HCC TME, focusing on M2 macrophages. Gene expression profiles were analysed to identify key molecules, with ATP6V1E1 as a primary focus. We employed Gene Set Enrichment Analysis (GSEA) and Kaplan-Meier survival analysis to investigate the molecular pathways and prognostic significance of ATP6V1E1. A prognostic model was developed using multivariate Cox regression analysis based on ATP6V1E1-related molecules, and functional impacts were assessed through cell proliferation assays. M2 macrophages were the dominant cell type in the HCC TME, significantly correlating with adverse survival outcomes. ATP6V1E1 was robustly associated with advanced disease stages and poor prognostic features such as vascular invasion and elevated alpha-fetoprotein levels. GSEA linked high ATP6V1E1 expression to critical oncogenic pathways, including immunosuppression and angiogenesis, and reduced activity in metabolic processes like bile acid and fatty acid metabolism. The prognostic model stratified HCC patients into distinct risk categories, showing high predictive accuracy (1-year AUC = 0.775, 3-year AUC = 0.709 and 5-year AUC = 0.791). In vitro assays demonstrated that ATP6V1E1 knockdown markedly inhibited the proliferation of HCC cells. The study underscores the significance of M2 macrophages and ATP6V1E1 in HCC, highlighting their potential as therapeutic and prognostic targets.

Exploring the multifaceted role of key lncRNA in glioma: From genetic expression to clinical implications and immunotherapy potential.

Background: Long non-coding RNAs (lncRNAs) are implicated in a variety of regulatory functions within tumors, yet their specific roles in glioma remain underexplored. Methods: We extracted glioma patient data from The Cancer Genome Atlas and UCSC Xena database for analysis using R, focusing on genomic characterization, biological enrichment, immune evaluation, and the development of a predictive model employing machine learning techniques. Additionally, we conducted cell culture and proliferation assays. Results: Our analysis revealed that the lncRNA SLC16A1-AS1 plays a pivotal role in glioma pathogenesis and prognosis. We observed that abnormal expression of SLC16A1-AS1 varied with tumor grade, IDH mutation status, and histological type, correlating with worse survival outcomes. Genomically, SLC16A1-AS1 was associated with Tumor Mutational Burden and other prognostic biomarkers. The expression of this lncRNA was also linked to the activation of critical biological pathways and appeared to modulate the immune microenvironment, enhancing the presence of immune cells and checkpoints, which may be predictive of immunotherapy outcomes. Our predictive model, constructed from genes associated with SLC16A1-AS1, accurately forecasted glioma prognosis, strongly correlating with survival and treatment responses. In vitro experiments further demonstrated that SLC16A1-AS1 significantly influences glioma cell proliferation, invasion, and migration, underscoring its role in tumor aggression and its potential as a therapeutic target. Conclusions: This study underscores the significant influence of SLC16A1-AS1 on glioma progression and prognosis, with its expression correlating with tumor traits and immune responses. The findings highlight the potential of targeting SLC16A1-AS1 in therapeutic strategies aimed at mitigating glioma aggressiveness.

Blockage of L2HGDH-mediated S-2HG catabolism orchestrates macrophage polarization to elicit antitumor immunity.

The high infiltration of tumor-associated macrophages (TAMs) in the immunosuppressive tumor microenvironment prominently attenuates the efficacy of immune checkpoint blockade (ICB) therapies, yet the underlying mechanisms are not fully understood. Here, we investigate the metabolic profile of TAMs and identify S-2-hydroxyglutarate (S-2HG) as a potential immunometabolite that shapes macrophages into an antitumoral phenotype. Blockage of L-2-hydroxyglutarate dehydrogenase (L2HGDH)-mediated S-2HG catabolism in macrophages promotes tumor regression. Mechanistically, based on its structural similarity to α-ketoglutarate (α-KG), S-2HG has the potential to block the enzymatic activity of 2-oxoglutarate-dependent dioxygenases (2-OGDDs), consequently reshaping chromatin accessibility. Moreover, S-2HG-treated macrophages enhance CD8+ T cell-mediated antitumor activity and sensitivity to anti-PD-1 therapy. Overall, our study uncovers the role of blockage of L2HGDH-mediated S-2HG catabolism in orchestrating macrophage antitumoral polarization and, further, provides the potential of repolarizing macrophages by S-2HG to overcome resistance to anti-PD-1 therapy.

Combined ROS Sensitive Folate Receptor Targeted Micellar Formulations of Curcumin Effective Against Rheumatoid Arthritis in Rat Model.

Introduction: Rheumatoid arthritis (RA) is an inflammatory immune-mediated disease that involves synovitis, cartilage destruction, and even joint damage. Traditional agents used for RA therapy remain unsatisfactory because of their low efficiency and obvious adverse effects. Therefore, we here established RA microenvironment-responsive targeted micelles that can respond to the increase in reactive oxygen species (ROS) levels in the joint and improve macrophage-specific targeting of loaded drugs. Methods: We here prepared ROS-responsive folate-modified curcumin micelles (TK-FA-Cur-Ms) in which thioketal (TK) was used as a ROS-responsive linker for modifying polyethylene glycol 5000 (PEG5000) on the micellar surface. When micelles were in the ROS-overexpressing inflammatory microenvironment, the PEG5000 hydration layer was shed, and the targeting ligand FA was exposed, thereby enhancing cellular uptake by macrophages through active targeting. The targeting, ROS sensitivity and anti-inflammatory properties of the micelles were assessed in vitro. Collagen-induced arthritis (CIA) rats model was utilized to investigate the targeting, expression of serum inflammatory factors and histology change of the articular cartilage by micelles in vivo. Results: TK-FA-Cur-Ms had a particle size of 90.07 ± 3.44 nm, which decreased to 78.87 ± 2.41 nm after incubation with H2O2. The micelles exhibited in vitro targeting of RAW264.7 cells and significantly inhibited inflammatory cytokine levels. Pharmacodynamic studies have revealed that TK-FA-Cur-Ms prolonged the drug circulation and exhibited augmented cartilage-protective and anti-inflammatory effects in vivo. Conclusion: The unique ROS-responsive targeted micelles with targeting, ROS sensitivity and anti-inflammatory properties were successfully prepared and may offer an effective therapeutic strategy against RA.

[Analysis of CHIP-Related Mutation and Risk of Cardio-Cerebrovasculars Events in Patients with Myeloproliferative Neoplasms].

OBJECTIVE: To analyze the mutant spectrum of clonal hematopoiesis of indeterminate potential (CHIP) related mutations and clinical characteristics and to explore the correlation and the possible mechanism between CHIP-related mutations and cardio-cerebrovasculars events (CCEs) in patients with myeloproliferative neoplasms (MPNs). METHODS: The clinical data and next-generation sequencing results of 73 MPN patients in Beijing Anzhen Hospital from August 2019 to July 2022 were retrospectively analyzed. Statistical analyses were conducted by multivariate logistic regression for the effects of CHIP-related mutations and inflammatory cytokines on CCEs for MPNs patients. RESULTS: Fifty-five cases of MPN (75.3%) showed positive in CHIP-related genes. There was no significant difference in variant allele frequency of CHIP-related gene between essential thrombocythemia (ET) and polycythemia vera (PV). CHIP-related gene mutations were mainly single gene mutations, with mutation rate from high to low as JAK2V617F (63.0%, 46/73), ASXL1 (16.4%, 12/73), TET2 (11.0%, 8/73), DNMT3A (9.6%, 7/73), SRSF2 (6.9%, 5/73), SF3B1 (4.1%, 3/73), TP53(1.4%, 1/73) and PPM1D (1.4%, 1/73). The mutation rate of CHIP-related genes in MPN patients >60 years old was significantly higher than that in the patients ≤60 years old ï¼»91.7%(33/36) vs 59.5%(22/37)ï¼½. CCEs occurred in 27 MPNs patients (37.0%, MPNs/CCEs), and 5 had recurrent CCEs, all of which were arterial events. Age (62.8±12.8 years vs 53.9±15.8 years, P =0.015), IL-1ß level (17.7±26.0 vs 4.3±8.6, P =0.012), IL-8 level (360.7±598.6 vs 108.3±317.0, P =0.045), the proportion of the patients with thrombosis history (29.6% vs 2.2%, P =0.020), and the detection rate of CHIP-related mutations (88.9% vs 67.4%, P =0.040) in the group with CCEs were higher than those in the group without CCEs. Multivariate Logistic regression analysis showed that age（OR =0.917， 95%CI ：0.843-0.999, P =0.047）, thrombosis history (OR =34.148， 95%CI ：2.392-487.535, P =0.009), any CHIP-related mutations(OR =16.065， 95%CI ：1.217-212.024， P =0.035), and elevated level of IL-1ß (OR =0.929， 95%CI ：0.870-0.992， P =0.027) were independent risk factors for MPNs/CCEs. CHIP-related gene mutations were not associated with CCEs in MPN patients, but DNMT3A (OR =88.717， 95%CI ：2.690-292.482， P =0.012) and ASXL1 (OR =7.941， 95%CI ：1.045-60.353， P =0.045) were independent risk factors for CCEs in PV. CONCLUSION: There is a higher mutation rate of CHIP-related genes in MPN patients, especially those over 60 years old. Older age, thrombosis history, CHIP-related mutations and IL-1ß elevated levels are independent risk factors for CCEs in MPN. DNMT3A and ASXL1 mutations are independent risk factors for CCEs in PV patients. CHIP-related gene mutations and inflammatory cytokine IL-1 ß elevated levels may be the novel risk factors for CCEs in MPN.

Impaired glycine neurotransmission causes adolescent idiopathic scoliosis.

Adolescent idiopathic scoliosis (AIS) is the most common form of spinal deformity, affecting millions of adolescents worldwide, but it lacks a defined theory of etiopathogenesis. Because of this, treatment of AIS is limited to bracing and/or invasive surgery after onset. Preonset diagnosis or preventive treatment remains unavailable. Here, we performed a genetic analysis of a large multicenter AIS cohort and identified disease-causing and predisposing variants of SLC6A9 in multigeneration families, trios, and sporadic patients. Variants of SLC6A9, which encodes glycine transporter 1 (GLYT1), reduced glycine-uptake activity in cells, leading to increased extracellular glycine levels and aberrant glycinergic neurotransmission. Slc6a9 mutant zebrafish exhibited discoordination of spinal neural activities and pronounced lateral spinal curvature, a phenotype resembling human patients. The penetrance and severity of curvature were sensitive to the dosage of functional glyt1. Administration of a glycine receptor antagonist or a clinically used glycine neutralizer (sodium benzoate) partially rescued the phenotype. Our results indicate a neuropathic origin for "idiopathic" scoliosis, involving the dysfunction of synaptic neurotransmission and central pattern generators (CPGs), potentially a common cause of AIS. Our work further suggests avenues for early diagnosis and intervention of AIS in preadolescents.

Structured diagnostic scheme clinical experience sharing: a prospective study of 320 cases of fever of unknown origin in a tertiary hospital in North China.

BACKGROUND: There has been little research on the long-term clinical outcomes of patients discharged due to undiagnosed fevers of unknown origin (FUO). The purpose of this study was to determine how fever of unknown origin (FUO) evolves over time and to determine the prognosis of patients in order to guide clinical diagnosis and treatment decisions. METHODS: Based on FUO structured diagnosis scheme, prospectively included 320 patients who hospitalized at the Department of Infectious Diseases of the Second Hospital of Hebei Medical University from March 15, 2016 to December 31,2019 with FUO, to analysis the cause of FUO, pathogenetic distribution and prognosis, and to compare the etiological distribution of FUO between different years, genders, ages, and duration of fever. RESULTS: Among the 320 patients, 279 were finally diagnosed through various types of examination or diagnostic methods, and the diagnosis rate was 87.2%. Among all the causes of FUO, 69.3% were infectious diseases, of which Urinary tract infection 12.8% and lung infection 9.7% were the most common. The majority of pathogens are bacteria. Among contagious diseases, brucellosis is the most common. Non-infectious inflammatory diseases were responsible for 6.3% of cases, of which systemic lupus erythematosus(SLE) 1.9% was the most common; 5% were neoplastic diseases; 5.3% were other diseases; and in 12.8% of cases, the cause was unclear. In 2018-2019, the proportion of infectious diseases in FUO was higher than 2016-2017 (P < 0.05). The proportion of infectious diseases was higher in men and older FUO than in women and young and middle-aged (P < 0.05). According to follow-up, the mortality rate of FUO patients during hospitalization was low at 1.9%. CONCLUSIONS: Infectious diseases are the principal cause of FUO. There are temporal differences in the etiological distribution of FUO, and the etiology of FUO is closely related to the prognosis. It is important to identify the etiology of patients with worsening or unrelieved disease.

Variants in the SOX9 transactivation middle domain induce axial skeleton dysplasia and scoliosis.

SOX9 is an essential transcriptional regulator of cartilage development and homeostasis. In humans, dysregulation of SOX9 is associated with a wide spectrum of skeletal disorders, including campomelic and acampomelic dysplasia, and scoliosis. The mechanism of how SOX9 variants contribute to the spectrum of axial skeletal disorders is not well understood. Here, we report four novel pathogenic variants of SOX9 identified in a large cohort of patients with congenital vertebral malformations. Three of these heterozygous variants are in the HMG and DIM domains, and for the first time, we report a pathogenic variant within the transactivation middle (TAM) domain of SOX9 . Probands with these variants exhibit variable skeletal dysplasia, ranging from isolated vertebral malformation to acampomelic dysplasia. We also generated a Sox9 hypomorphic mutant mouse model bearing a microdeletion within the TAM domain ( Sox9 Asp272del ). We demonstrated that disturbance of the TAM domain with missense mutation or microdeletion results in reduced protein stability but does not affect the transcriptional activity of SOX9. Homozygous Sox9 Asp272del mice exhibited axial skeletal dysplasia including kinked tails, ribcage anomalies, and scoliosis, recapitulating phenotypes observed in human, while heterozygous mutants display a milder phenotype. Analysis of primary chondrocytes and the intervertebral discs in Sox9 Asp272del mutant mice revealed dysregulation of a panel of genes with major contributions of the extracellular matrix, angiogenesis, and ossification-related processes. In summary, our work identified the first pathologic variant of SOX9 within the TAM domain and demonstrated that this variant is associated with reduced SOX9 protein stability. Our finding suggests that reduced SOX9 stability caused by variants in the TAM domain may be responsible for the milder forms of axial skeleton dysplasia in humans.

Ten-year trends in surgical management of 1207 congenital scoliosis.

PURPOSE: To report and analyze development trends in the surgical treatment of congenital scoliosis (CS) in a large CS cohort over a 10-year period. METHODS: We retrospectively searched and extracted medical records of CS inpatients receiving posterior instrumented fusion surgery at our institute from January 2010 to December 2019. We analyzed information on demographics and surgical information, including the surgical approach, number of fused segments, use of osteotomy and titanium cage implantation, length of stay, intraoperative blood loss, and rates of complications and readmission. RESULTS: 1207 CS inpatients were included. In the past decade, the proportion of patients younger than 5 years increased from 15.5 to 26.9%. The average number of fused segments decreased from 9.24 to 7.48, and the proportion of patients treated with short-segment fusion increased from 13.4 to 30.3%. The proportion of patients treated with osteotomy and titanium cage implantation increased from 55.65% and 12.03% to 76.5% and 40.22%. The average length of stay and blood loss decreased from 16.5 days and 816.1 ml to 13.5 days and 501.7 ml. The complication and readmission rates also decreased during these ten years. CONCLUSION: During this ten-year period, the surgical treatment of CS at our institute showed trends toward a younger age at fusion, lower number of fused segments, higher rate of osteotomy and titanium cage implantation, reduced blood loss, shorter length of stay and lower rate complications and readmission. These results suggest performing osteotomy combined with titanium cage implantation at an earlier age can achieve fewer fused segments and complications.

HuR Promotes the Differentiation of Goat Skeletal Muscle Satellite Cells by Regulating Myomaker mRNA Stability.

Human antigen R (HuR) is an RNA-binding protein that contributes to a wide variety of biological processes and diseases. HuR has been demonstrated to regulate muscle growth and development, but its regulatory mechanisms are not well understood, especially in goats. In this study, we found that HuR was highly expressed in the skeletal muscle of goats, and its expression levels changed during longissimus dorsi muscle development in goats. The effects of HuR on goat skeletal muscle development were explored using skeletal muscle satellite cells (MuSCs) as a model. The overexpression of HuR accelerated the expression of myogenic differentiation 1 (MyoD), Myogenin (MyoG), myosin heavy chain (MyHC), and the formation of myotubes, while the knockdown of HuR showed opposite effects in MuSCs. In addition, the inhibition of HuR expression significantly reduced the mRNA stability of MyoD and MyoG. To determine the downstream genes affected by HuR at the differentiation stage, we conducted RNA-Seq using MuSCs treated with small interfering RNA, targeting HuR. The RNA-Seq screened 31 upregulated and 113 downregulated differentially expressed genes (DEGs) in which 11 DEGs related to muscle differentiation were screened for quantitative real-time PCR (qRT-PCR) detection. Compared to the control group, the expression of three DEGs (Myomaker, CHRNA1, and CAPN6) was significantly reduced in the siRNA-HuR group (p < 0.01). In this mechanism, HuR bound to Myomaker and increased the mRNA stability of Myomaker. It then positively regulated the expression of Myomaker. Moreover, the rescue experiments indicated that the overexpression of HuR may reverse the inhibitory impact of Myomaker on myoblast differentiation. Together, our findings reveal a novel role for HuR in promoting muscle differentiation in goats by increasing the stability of Myomaker mRNA.

Retrospective Analysis of Associated Anomalies in 636 Patients with Operatively Treated Congenital Scoliosis.

BACKGROUND: Congenital scoliosis is frequently associated with anomalies in multiple organ systems. However, the prevalence and distribution of associated anomalies remain unclear, and there is a large amount of variation in data among different studies. METHODS: Six hundred and thirty-six Chinese patients who had undergone scoliosis correction surgery at Peking Union Medical College Hospital from January 2012 to July 2019 were recruited, as a part of the Deciphering disorders Involving Scoliosis and COmorbidities (DISCO) study. The medical data for each subject were collected and analyzed. RESULTS: The mean age (and standard deviation) at the time of presentation for scoliosis was 6.4 ± 6.3 years, and the mean Cobb angle of the major curve was 60.8° ± 26.5°. Intraspinal abnormalities were found in 186 (30.3%) of 614 patients, with diastematomyelia being the most common anomaly (59.1%; 110 of 186). The prevalence of intraspinal abnormalities was remarkably higher in patients with failure of segmentation and mixed deformities than in patients with failure of formation (p < 0.001). Patients with intraspinal anomalies showed more severe deformities, including larger Cobb angles of the major curve (p < 0.001). We also demonstrated that cardiac anomalies were associated with remarkably worse pulmonary function, i.e., lower forced expiratory volume in the first second (FEV1), forced vital capacity (FVC), and peak expiratory flow (PEF). Additionally, we identified associations among different concomitant malformations. We found that patients with musculoskeletal anomalies of types other than intraspinal and maxillofacial were 9.2 times more likely to have additional maxillofacial anomalies. CONCLUSIONS: In our cohort, comorbidities associated with congenital scoliosis occurred at a rate of 55%. To our knowledge, our study is the first to show that patients with congenital scoliosis and cardiac anomalies have reduced pulmonary function, as demonstrated by lower FEV1, FVC, and PEF. Moreover, the potential associations among concomitant anomalies revealed the importance of a comprehensive preoperative evaluation scheme. LEVEL OF EVIDENCE: Diagnostic Level III. See Instructions for Authors for a complete description of levels of evidence.

Landscape of Secondary Findings in Chinese Population: A Practice of ACMG SF v3.0 List.

Clinical exome sequencing (CES) has shown great utility in the diagnosis of Mendelian disorders. CES can unravel secondary findings (SFs) unrelated to the primary diagnosis but with potential health implications. The American College of Medical Genetics and Genomics (ACMG) has published a guideline for reporting secondary findings and recently updated an ACMG SF v3.0 list comprising 73 genes. Several studies have been performed to explore the prevalence of SFs. However, the data were limited in the Chinese population. In this study, we evaluated the genetic data of 2987 individuals from the Deciphering Disorders Involving Scoliosis and COmorbidities (DISCO) study group in accordance with the ACMG SF v3.0 list. The detected variants were evaluated using the ACMG classification guidelines, HGMD, and ClinVar database. Totally, 157 (157/2987, 5.3%) individuals had reportable variants within genes associated with cancer, cardiovascular, metabolic, and miscellaneous phenotypes. We identified 63 known pathogenic (KP) variants in 72 individuals (72/2987, 2.4%) and 96 expected pathogenic (EP) variants in 105 individuals (3.5%). Forty-five individuals carried SFs in v3.0 newly added genes, which accounted for 1.5% of our cohort. Our findings could contribute to existing knowledge of secondary findings in different ethnicities and indicate the necessity for clinicians to update the SFs gene list.

TBX6 as a cause of a combined skeletal-kidney dysplasia syndrome.

TBX6 encodes transcription-factor box 6, a transcription factor critical to paraxial mesoderm segmentation and somitogenesis during embryonic development. TBX6 haploinsufficiency is believed to drive the skeletal and kidney phenotypes associated with the 16p11.2 deletion syndrome. Heterozygous and biallelic variants in TBX6 are associated with vertebral and rib malformations (TBX6-associated congenital scoliosis) and spondylocostal dysostosis, and heterozygous TBX6 variants are associated with increased risk of genitourinary tract malformations. Combined skeletal and kidney phenotypes in individuals harboring heterozygous or biallelic TBX6 variants are rare. Here, we present seven individuals with vertebral and rib malformations and structural kidney differences associated with heterozygous TBX6 gene deletion in trans with a hypomorphic TBX6 allele or biallelic TBX6 variants. Our case series highlights the association between TBX6 and both skeletal and kidney disease.

Mitochondrial PKM2 deacetylation by procyanidin B2-induced SIRT3 upregulation alleviates lung ischemia/reperfusion injury.

Apoptosis is a critical event in the pathogenesis of lung ischemia/reperfusion (I/R) injury. Sirtuin 3 (SIRT3), an important deacetylase predominantly localized in mitochondria, regulates diverse physiological processes, including apoptosis. However, the detailed mechanisms by which SIRT3 regulates lung I/R injury remain unclear. Many polyphenols strongly regulate the sirtuin family. In this study, we found that a polyphenol compound, procyanidin B2 (PCB2), activated SIRT3 in mouse lungs. Due to this effect, PCB2 administration attenuated histological lesions, relieved pulmonary dysfunction, and improved the survival rate of the murine model of lung I/R injury. Additionally, this treatment inhibited hypoxia/reoxygenation (H/R)-induced A549 cell apoptosis and rescued Bcl-2 expression. Using Sirt3-knockout mice and specific SIRT3 knockdown in vitro, we further found that SIRT3 strongly protects against lung I/R injury. Sirt3 deficiency or enzymatic inactivation substantially aggravated lung I/R-induced pulmonary lesions, promoted apoptosis, and abolished PCB2-mediated protection. Mitochondrial pyruvate kinase M2 (PKM2) inhibits apoptosis by stabilizing Bcl-2. Here, we found that PKM2 accumulates and is hyperacetylated in mitochondria upon lung I/R injury. By screening the potential sites of PKM2 acetylation, we found that SIRT3 deacetylates the K433 residue of PKM2 in A549 cells. Transfection with a deacetylated mimic plasmid of PKM2 noticeably reduced apoptosis, while acetylated mimic transfection abolished the protective effect of PKM2. Furthermore, PKM2 knockdown or inhibition in vivo significantly abrogated the antiapoptotic effects of SIRT3 upregulation. Collectively, this study provides the first evidence that the SIRT3/PKM2 pathway is a protective target for the suppression of apoptosis in lung I/R injury. Moreover, this study identifies K433 deacetylation of PKM2 as a novel modification that regulates its anti-apoptotic activity. In addition, PCB2-mediated modulation of the SIRT3/PKM2 pathway may significantly protect against lung I/R injury, suggesting a novel prophylactic strategy for lung I/R injury.

Phenotype expansion of variants affecting p38 MAPK signaling in hypospadias patients.

BACKGROUND: Hypospadias is a congenital anomaly of the male urogenital system. Genetics factors play an important role in its pathogenesis. To search for potential causal genes/variants for hypospadias, we performed exome sequencing in a pedigree with three patients across two generations and a cohort of 49 sporadic patients with hypospadias. RESULTS: A novel BRAF variant (NM_004333.6: c.362C > A) was found to co-segregate with the hypospadias phenotype in the disease pedigree. In cells overexpressing the BRAF mutant, the phosphorylation level of p38 MAPK was significantly increased as compared with the cells overexpressing the wild-type BRAF or RASopathy-related BRAF mutant. This variant further led to a reduced transcription level of the SRY gene, which is essential for the normal development of the male reproductive system. In the cohort of sporadic patients, we identified two additional variants in p38 MAPK signaling-related genes (TRIM67 and DAB2IP) potentially associated with hypospadias. CONCLUSION: Our study expands the phenotypic spectrum of variants affecting p38 MAPK signaling toward the involvement of hypospadias.

NRF2 drives an oxidative stress response predictive of breast cancer.

Many breast cancer patients are diagnosed with small, well-differentiated, hormone receptor-positive tumors. Risk of relapse is not easily identified in these patients, resulting in overtreatment. To identify metastasis-related gene expression patterns, we compared the transcriptomes of the non-metastatic 67NR and metastatic 66cl4 cell lines from the murine 4T1 mammary tumor model. The transcription factor nuclear factor, erythroid 2-like 2 (NRF2, encoded by NFE2L2) was constitutively activated in the metastatic cells and tumors, and correspondingly a subset of established NRF2-regulated genes was also upregulated. Depletion of NRF2 increased basal levels of reactive oxygen species (ROS) and severely reduced ability to form primary tumors and lung metastases. Consistently, a set of NRF2-controlled genes was elevated in breast cancer biopsies. Sixteen of these were combined into a gene expression signature that significantly improves the PAM50 ROR score, and is an independent, strong predictor of prognosis, even in hormone receptor-positive tumors.

Delineation of dual molecular diagnosis in patients with skeletal deformity.

BACKGROUND: Skeletal deformity is characterized by an abnormal anatomical structure of bone and cartilage. In our previous studies, we have found that a substantial proportion of patients with skeletal deformity could be explained by monogenic disorders. More recently, complex phenotypes caused by more than one genetic defect (i.e., dual molecular diagnosis) have also been reported in skeletal deformities and may complicate the diagnostic odyssey of patients. In this study, we report the molecular and phenotypic characteristics of patients with dual molecular diagnosis and variable skeletal deformities. RESULTS: From 1108 patients who underwent exome sequencing, we identified eight probands with dual molecular diagnosis and variable skeletal deformities. All eight patients had dual diagnosis consisting of two autosomal dominant diseases. A total of 16 variants in 12 genes were identified, 5 of which were of de novo origin. Patients with dual molecular diagnosis presented blended phenotypes of two genetic diseases. Mendelian disorders occurred more than once include Osteogenesis Imperfecta Type I (COL1A1, MIM:166200), Neurofibromatosis, Type I (NF1, MIM:162200) and Marfan Syndrome (FBN1, MIM:154700). CONCLUSIONS: This study demonstrated the complicated skeletal phenotypes associated with dual molecular diagnosis. Exome sequencing represents a powerful tool to detect such complex conditions.

Exome-wide Analysis of De Novo and Rare Genetic Variants in Patients With Brain Arteriovenous Malformation.

BACKGROUND AND OBJECTIVES: Brain arteriovenous malformation (bAVM) is a congenital disorder and a leading cause of hemorrhagic stroke. Germline genetic variants play an essential role in the pathogenesis of bAVM. However, the biological relevance of disease-associated genes identified in previous studies is elusive. In this study, we aim to systematically investigate the contribution of germline variants to bAVM and explore the critical molecular pathways underlying the pathogenesis of bAVM. METHODS: Probands with sporadic bAVM were consecutively recruited into this study from November 2015 to November 2018 and underwent exome sequencing. The controls were aggregated from individuals who were not known to have vascular malformation and underwent exome sequencing for clinical or research purposes. The retained control dataset included 4,609 individuals, including 251 individuals with parental samples sequenced. We first analyzed de novo variants in cases and controls and performed a pathway enrichment analysis. A gene-based rare variant association analysis was then performed to identify genes whose variants were significantly enriched in cases. RESULTS: We collected an exome-sequenced bAVM cohort consisting of 152 trios and 40 singletons. By first focusing on de novo variants, we observed a significant mutational burden of likely gene-disrupting variants in cases vs controls. By performing a pathway enrichment analysis of all nonsynonymous de novo variants identified in cases, we found the angiopoietin-like protein 8 (ANGPTL8) regulatory pathway to be significantly enriched in patients with bAVM. Through an exome-wide rare variant association analysis utilizing 4,394 in-house exome data as controls, we identified SLC19A3 as a disease-associated gene for bAVM. In addition, we found that the SLC19A3 variants in cases are preferably located at the N' side of the SLC19A3 protein. These findings implicate a phenotypic expansion of SLC19A3-related disorders with a domain-specific effect. DISCUSSION: This study provides insights into the biological basis of bAVM by identifying novel molecular pathways and candidate genes.

DrABC: deep learning accurately predicts germline pathogenic mutation status in breast cancer patients based on phenotype data.

BACKGROUND: Identifying breast cancer patients with DNA repair pathway-related germline pathogenic variants (GPVs) is important for effectively employing systemic treatment strategies and risk-reducing interventions. However, current criteria and risk prediction models for prioritizing genetic testing among breast cancer patients do not meet the demands of clinical practice due to insufficient accuracy. METHODS: The study population comprised 3041 breast cancer patients enrolled from seven hospitals between October 2017 and 11 August 2019, who underwent germline genetic testing of 50 cancer predisposition genes (CPGs). Associations among GPVs in different CPGs and endophenotypes were evaluated using a case-control analysis. A phenotype-based GPV risk prediction model named DNA-repair Associated Breast Cancer (DrABC) was developed based on hierarchical neural network architecture and validated in an independent multicenter cohort. The predictive performance of DrABC was compared with currently used models including BRCAPRO, BOADICEA, Myriad, PENN II, and the NCCN criteria. RESULTS: In total, 332 (11.3%) patients harbored GPVs in CPGs, including 134 (4.6%) in BRCA2, 131 (4.5%) in BRCA1, 33 (1.1%) in PALB2, and 37 (1.3%) in other CPGs. GPVs in CPGs were associated with distinct endophenotypes including the age at diagnosis, cancer history, family cancer history, and pathological characteristics. We developed a DrABC model to predict the risk of GPV carrier status in BRCA1/2 and other important CPGs. In predicting GPVs in BRCA1/2, the performance of DrABC (AUC = 0.79 [95% CI, 0.74-0.85], sensitivity = 82.1%, specificity = 63.1% in the independent validation cohort) was better than that of previous models (AUC range = 0.57-0.70). In predicting GPVs in any CPG, DrABC (AUC = 0.74 [95% CI, 0.69-0.79], sensitivity = 83.8%, specificity = 51.3% in the independent validation cohort) was also superior to previous models in their current versions (AUC range = 0.55-0.65). After training these previous models with the Chinese-specific dataset, DrABC still outperformed all other methods except for BOADICEA, which was the only previous model with the inclusion of pathological features. The DrABC model also showed higher sensitivity and specificity than the NCCN criteria in the multi-center validation cohort (83.8% and 51.3% vs. 78.8% and 31.2%, respectively, in predicting GPVs in any CPG). The DrABC model implementation is available online at http://gifts.bio-data.cn/ . CONCLUSIONS: By considering the distinct endophenotypes associated with different CPGs in breast cancer patients, a phenotype-driven prediction model based on hierarchical neural network architecture was created for identification of hereditary breast cancer. The model achieved superior performance in identifying GPV carriers among Chinese breast cancer patients.