Hyperhomocysteinemia lowers serum testosterone concentration via impairing testosterone production in Leydig cells.

Hyperhomocysteinemia (HHcy) plays a salient role in male infertility. However, whether HHcy interferes with testosterone production remains inconclusive. Here, we reported a lower serum testosterone level in HHcy mice. Single-cell RNA sequencing revealed that genes related to testosterone biosynthesis, together with nuclear receptor subfamily 5 group A member 1 (Nr5a1), a key transcription factor for steroidogenic genes, were downregulated in the Leydig cells (LCs) of HHcy mice. Mechanistically, Hcy lowered trimethylation of histone H3 on lysine 4 (H3K4me3), which was bound on the promoter region of Nr5a1, resulting in downregulation of Nr5a1. Intriguingly, we identified an unknown cell cluster annotated as Macrophage-like Leydig cells (McLCs), expressing both LCs and macrophages markers. In HHcy mice, McLCs were shifted toward pro-inflammatory phenotype and thus promoted inflammatory response in LC. Betaine supplementation rescued the downregulation of NR5A1 and restored the serum testosterone level in HHcy mice. Overall, our study highlights an etiological role of HHcy in LCs dysfunction.

AutoCNV: a semiautomatic CNV interpretation system based on the 2019 ACMG/ClinGen Technical Standards for CNVs.

BACKGROUND: The American College of Medical Genetics and Genomics (ACMG) and the Clinical Genome Resource (ClinGen) presented technical standards for interpretation and reporting of constitutional copy-number variants in 2019 (the standards). Although ClinGen developed a web-based CNV classification calculator based on scoring metrics, it can only track and tally points that have been assigned based on observed evidence. Here, we developed AutoCNV (a semiautomatic automated CNV interpretation system) based on the standards, which can automatically generate predictions on 18 and 16 criteria for copy number loss and gain, respectively. RESULTS: We assessed the performance of AutoCNV using 72 CNVs evaluated by external independent reviewers and 20 illustrative case examples. Using AutoCNV, it showed that 100 % (72/72) and 95 % (19/20) of CNVs were consistent with the reviewers' and ClinGen-verified classifications, respectively. AutoCNV only required an average of less than 5 milliseconds to obtain the result for one CNV with automated scoring. We also applied AutoCNV for the interpretation of CNVs from the ClinVar database and the dbVar database. We also developed a web-based version of AutoCNV (wAutoCNV). CONCLUSIONS: AutoCNV may serve to assist users in conducting in-depth CNV interpretation, to accelerate and facilitate the interpretation process of CNVs and to improve the consistency and reliability of CNV interpretation.

dbInDel: a database of enhancer-associated insertion and deletion variants by analysis of H3K27ac ChIP-Seq.

SUMMARY: Cancer hallmarks rely on its specific transcriptional programs, which are dysregulated by multiple mechanisms, including genomic aberrations in the DNA regulatory regions. Genome-wide association studies have shown many variants are found within putative enhancer elements. To provide insights into the regulatory role of enhancer-associated non-coding variants in cancer epigenome, and to facilitate the identification of functional non-coding mutations, we present dbInDel, a database where we have comprehensively analyzed enhancer-associated insertion and deletion variants for both human and murine samples using ChIP-Seq data. Moreover, we provide the identification and visualization of upstream TF binding motifs in InDel-containing enhancers. Downstream target genes are also predicted and analyzed in the context of cancer biology. The dbInDel database promotes the investigation of functional contributions of non-coding variants in cancer epigenome. AVAILABILITY AND IMPLEMENTATION: The database, dbInDel, can be accessed from http://enhancer-indel.cam-su.org/. SUPPLEMENTARY INFORMATION: Supplementary data are available at Bioinformatics online.

dbCoRC: a database of core transcriptional regulatory circuitries modeled by H3K27ac ChIP-seq signals.

Core transcription regulatory circuitry (CRC) is comprised of a small group of self-regulated transcription factors (TFs) and their interconnected regulatory loops. Studies from embryonic stem cells and other cellular models have revealed the elementary roles of CRCs in transcriptional control of cell identity and cellular fate. Systematic identification and subsequent archiving of CRCs across diverse cell types and tissues are needed to explore both cell/tissue type-specific and disease-associated transcriptional networks. Here, we present a comprehensive and interactive database (dbCoRC, http://dbcorc.cam-su.org) of CRC models which are computationally inferred from mapping of super-enhancer and prediction of TF binding sites. The current version of dbCoRC contains CRC models for 188 human and 50 murine cell lines/tissue samples. In companion with CRC models, this database also provides: (i) super enhancer, typical enhancer, and H3K27ac landscape for individual samples, (ii) putative binding sites of each core TF across the super-enhancer regions within CRC and (iii) expression of each core TF in normal or cancer cells/tissues. The dbCoRC will serve as a valuable resource for the scientific community to explore transcriptional control and regulatory circuitries in biological processes related to, but not limited to lineage specification, tissue homeostasis and tumorigenesis.

Urinary angiotensinogen: an indicator of active antineutrophil cytoplasmic antibody-associated glomerulonephritis.

BACKGROUND: One of the major challenges in improving the management of antineutrophil cytoplasmic antibody-associated glomerulonephritis (ANCA-GN) is the lack of a disease-specific indicator for histological lesions and disease activity. Here we tested the utility of urinary angiotensinogen (UAGT) as a biomarker of renal disease activity in ANCA-GN. METHODS: A prospective, two-stage cohort study was performed in ANCA-GN patients. In Stage I, UAGT was measured at the time of renal biopsy in 69 patients from two centers (test set) and 25 patients from two other centers (validation set). In Stage II, UAGT was monitored in 50 subjects in the test set for 24 months. RESULTS: In Stage I, UAGT significantly increased in ANCA-GN patients, correlating well with cellular crescents formation and active interstitial inflammation. Patients with crescentic ANCA-GN exhibited the highest UAGT compared with other histopathological classes of ANCA-GN. After multivariable adjustment, the highest quartile of UAGT, compared with the lowest quartile, associated with a 6-fold increased risk of crescentic ANCA-GN. For predicting crescentic ANCA-GN, UAGT [area under the receiver operating characteristics curve (AUC) = 0.88] outperformed albuminuria (AUC = 0.73) and estimated glomerular filtration rate (AUC = 0.69). UAGT improved the performance of those clinical markers in diagnosing crescentic ANCA-GN (P < 0.034), suggesting a role of UAGT in identifying active crescentic ANCA-GN. In Stage II, UAGT decreased after immunotherapy and increased at the time of renal relapse during the 2-year follow-up, suggesting the usefulness of UAGT to monitor disease activity over time. CONCLUSIONS: These results suggest the potential use of UAGT for assessing disease activity and renal relapse in ANCA-GN.

Overexpression of Intrarenal Renin-Angiotensin System in Human Acute Tubular Necrosis.

BACKGROUND/AIMS: Acute tubular necrosis (ATN), a leading cause of acute kidney injury (AKI), is associated with decreased survival and increased progression of chronic kidney disease. A barrier to improving the clinical outcomes is the incomplete understanding of the pathogenesis of AKI. Our objective is to test the hypothesis that intrarenal renin-angiotensin system (RAS) is overexpressed in patients with ATN and could be an indicator of ATN severity. METHODS: A transversal study was conducted in patients with biopsy-proven ATN. Intrarenal expression of angiotensinogen and angiotensin II, and urinary angiotensinogen were measured. RESULTS: Patients with ATN demonstrated upregulation of intrarenal RAS, evidenced by upregulation of intrarenal angiotensinogen and angiotensin II. Patients with ATN also have elevated urinary angiotensinogen level that correlated with the overexpressed intrarenal RAS. Moreover, this increase in intrarenal RAS expression and urinary angiotensinogen was associated with the extent of acute tubular injury and urinary albumin excretion, respectively. CONCLUSIONS: We demonstrate that the intrarenal RAS is upregulated in patients with ATN and is associated with the severity of ATN. Urinary angiotensinogen reflects intrarenal RAS status, and is of value to assess the severity of ATN.

Advances in sequencing-based studies of microDNA and ecDNA: Databases, identification methods, and integration with single-cell analysis.

Extrachromosomal circular DNA (eccDNA) is a class of circular DNA molecules that originate from genomic DNA but are separate from chromosomes. They are common in various organisms, with sizes ranging from a few hundred to millions of base pairs. A special type of large extrachromosomal DNA (ecDNA) is prevalent in cancer cells. Research on ecDNA has significantly contributed to our comprehension of cancer development, progression, evolution, and drug resistance. The use of next-generation (NGS) and third-generation sequencing (TGS) techniques to identify eccDNAs throughout the genome has become a trend in current research. Here, we briefly review current advances in the biological mechanisms and applications of two distinct types of eccDNAs: microDNA and ecDNA. In addition to presenting available identification tools based on sequencing data, we summarize the most recent efforts to integrate ecDNA with single-cell analysis and put forth suggestions to promote the process.

Circlehunter: a tool to identify extrachromosomal circular DNA from ATAC-Seq data.

In cancer, extrachromosomal circular DNA (ecDNA), or megabase-pair amplified circular DNA, plays an essential role in intercellular heterogeneity and tumor cell revolution because of its non-Mendelian inheritance. We developed circlehunter ( https://github.com/suda-huanglab/circlehunter ), a tool for identifying ecDNA from ATAC-Seq data using the enhanced chromatin accessibility of ecDNA. Using simulated data, we showed that circlehunter has an F1 score of 0.93 at 30× local depth and read lengths as short as 35 bp. Based on 1312 ecDNAs predicted from 94 publicly available datasets of ATAC-Seq assays, we found 37 oncogenes contained in these ecDNAs with amplification characteristics. In small cell lung cancer cell lines, ecDNA containing MYC leads to amplification of MYC and cis-regulates the expression of NEUROD1, resulting in an expression pattern consistent with the NEUROD1 high expression subtype and sensitive to Aurora kinase inhibitors. This showcases that circlehunter could serve as a valuable pipeline for the investigation of tumorigenesis.

Identification of a novel eighteen-gene signature of recurrent metastasis neuroblastoma.

Neuroblastoma is the most common malignant tumor in childhood, and metastases occur in more than 30% patients. Recurrent metastasis is the main cause of poor prognosis and high mortality in neuroblastoma. In this regard, there is still a lack of sufficient biomarkers and effective therapies. Therefore, we performed a multi-omics analysis of neuroblastoma patients from Therapeutically Applicable Research To Generate Effective Treatments (TARGET). With clinical relapse site information, tumor samples derived from the primary site were divided into recurrent metastasis and primary tumor groups. The initial gene signature was obtained by comparing RNA-Seq and copy number variation differences. Survival data was used to further filter prognosis-related genes. This 18-gene signature consists of three clusters: tumor suppression, cell proliferation, and immunity. A super enhancer is involved in the enhanced expression of NCAPG in cluster2 together with IRF3. Based on the gene signature expression in primary neuroblastoma, it is possible to predict tumor metastasis before it occurs. According to the anticancer drug dataset of Genomics of Drug Sensitivity in Cancer (GDSC), vinorelbine and docetaxel were predicted to have high sensitivity against recurrent metastatic neuroblastoma. In conclusion, our study offers a novel metastasis biomarker and helps understand the mechanisms of tumor recurrent metastasis. KEY MESSAGES: We identified a novel eighteen-gene signature of recurrent metastasis neuroblastoma and build risk and classification models. We dissected the regulatory role of NCAPG in signatures. We found immune exhaustion and immunosuppression in recurrent metastasis neuroblastoma. Vinorelbine and docetaxel were predicted to have high sensitivity against recurrent metastatic neuroblastoma.

Urinary albumin-to-creatinine ratio and the risk of first stroke in Chinese hypertensive patients treated with angiotensin-converting enzyme inhibitors.

We aimed to evaluate the relationship of the albumin-to-creatinine ratio (ACR) with the risk of first stroke and examine possible effect modifiers in hypertensive patients. A total of 11,632 hypertensive participants with urinary ACR measurements and without a history of stroke from the China Stroke Primary Prevention Trial (CSPPT) were included in this analysis. The primary outcome was first stroke. Over a median follow-up of 4.4 years, 728 first strokes were identified, of which 633 were ischemic, 89 were hemorrhagic, and 6 were uncertain types. Overall, there was a significant positive association between natural log-transformed ACR and the risk of first stroke (HR, 1.11; 95% CI: 1.03-1.20) and first ischemic stroke (HR, 1.12; 95% CI: 1.03-1.22). Consistently, participants with ACR ≥ 10 mg/g had a significantly higher risk of first stroke (HR, 1.26; 95% CI: 1.06-1.50) and first ischemic stroke (HR, 1.33; 95% CI: 1.10-1.59) than those with ACR < 10 mg/g. Moreover, the association of ACR with first stroke was significantly stronger in participants with higher total homocysteine (tHcy) levels (<10 versus ≥ 10 µmol/L; P for interaction = 0.044). However, there was no significant association between ACR and first hemorrhagic stroke (per natural log [ACR] increment: HR, 1.02; 95% CI: 0.82-1.27). In summary, hypertensive patients with ACR ≥ 10 mg/g had a significantly increased risk of first stroke or first ischemic stroke. This positive association was more pronounced among participants with higher tHcy levels.

Urinary Matrix Metalloproteinase 7 Activated by Oxidative Stress Predicts Kidney Prognosis in Myeloperoxidase-Antineutrophil Cytoplasmic Antibody-Associated Vasculitis.

Aims: Effective and applicable predictors of end-stage kidney disease (ESKD) are needed for patients with myeloperoxidase-antineutrophil cytoplasmic antibody-associated vasculitis (MPO-AAV) and kidney involvement. We investigated whether urinary matrix metalloproteinase-7 (uMMP7) was associated with kidney injury severity and incident ESKD in MPO-AAV. Results: A prospective two-stage study was conducted in 150 patients with newly diagnosed MPO-AAV in two independent cohorts. uMMP7 was measured on the days of initial and repeat kidney biopsies. In stage I, a higher initial uMMP7 level was associated with a lower estimated glomerular filtration rate (eGFR), higher level of proteinuria, and greater extent of kidney pathologic lesions. This elevated uMMP7 protein level is activated and potentially derived from the enhanced kidney production induced by oxidative stress. In stage II, uMMP7 at initial biopsy was independently associated with the incidence of ESKD over 6 years. The higher uMMP7 group (vs. lower) had an adjusted hazard ratio of 3.79 (95% confidence interval [CI], 1.49-6.09) for ESKD in the test cohort. Findings were similar in the validation cohort. A combination of data from the two cohorts revealed that adding uMMP7 into clinical or clinicopathologic models significantly improved risk discrimination for future ESKD. Innovation: An elevated uMMP7 level in MPO-AAV was independently associated with severe kidney injury and incident ESKD. Conclusions: uMMP7 in MPO-AAV improves identification of patients at risk of ESKD and may enable early and optimized therapy to improve outcomes. Antioxid. Redox Signal. 37, 246-256.

Association of Urinary Matrix Metalloproteinase 7 Levels With Incident Renal Flare in Lupus Nephritis.

OBJECTIVE: Flares of lupus nephritis (LN) are frequent and associated with impaired renal prognosis. One major management obstacle in LN flare is the lack of effective methods to identify at-risk patients earlier in their disease course. This study was undertaken to test the utility of measurement of urinary matrix metalloproteinase 7 (MMP-7) for the dynamic surveillance of renal disease activity and prediction of renal flares in LN. METHODS: A prospective, 2-stage cohort study was performed in patients with LN. Urinary MMP-7 levels at the time of biopsy were evaluated in 154 patients with newly diagnosed LN in 2 independent cohorts. Urinary MMP-7 levels were assessed for correlation with renal histologic activity. Furthermore, after a minimum period of 12 months of renal disease remission, urinary MMP-7 levels were monitored bimonthly for 2 years in 65 patients with LN. The association between urinary MMP-7 levels and development of LN flare was analyzed. RESULTS: Urinary MMP-7 levels were elevated in patients with LN. A higher urinary MMP-7 level in LN was associated with greater renal histologic activity. As a marker for identifying LN patients with more severe renal histologic activity (i.e., a histologic activity index of ≥7), the level of urinary MMP-7 outperformed other clinical markers and improved their predictive performance, thus linking urinary MMP-7 levels to renal disease activity. Furthermore, among patients who had follow-up measurements of urinary MMP-7 after achievement of long-term remission of renal disease activity, an elevated urinary MMP-7 level during follow-up was independently associated with an increased risk of LN flare. This elevation in the urinary MMP-7 level hinted at the risk of an LN flare at an earlier time point prior to indications using conventional laboratory measures. Thus, use of the urinary MMP-7 level in conjunction with other clinical measures improved the prognostic value for prediction of an LN flare. CONCLUSION: Urinary MMP-7 levels in LN are correlated with renal histologic activity. An elevated urinary MMP-7 level detected after achievement of long-term renal disease remission is associated with a higher risk of incident renal flare in patients with LN.

Association of visit-to-visit variability in blood pressure and first stroke risk in hypertensive patients with chronic kidney disease.

OBJECTIVE: Data on the association between visit-to-visit variability (VVV) in blood pressure (BP) and the risk of stroke among hypertensive patients with chronic kidney disease (CKD) is limited. We aimed to evaluate the relation of VVV in BP with the risk of stroke, and examine any possible effect modifiers in hypertensive patients with mild-to-moderate CKD. METHODS: This is a post-hoc analysis of the China Stroke Primary Prevention Trial. A total of 3091 patients with estimated glomerular filtration rate 30-60 ml/min per 1.73 m and/or proteinuria at baseline, without occurring stroke and with BP measurements of at least two visits from randomization to the 12-month visit were included. The main VVV in BP was expressed as SD. The primary outcome was first stroke. RESULTS: The median subsequent treatment duration was 3.7 years. After multivariable adjustment, including baseline SBP and mean SBP during the first 12-month follow-up, there was a significantly positive relationship of SD SBP with the risk of subsequent first stroke (per SD increment; odds ratio, 1.41; 95% confidence interval: 1.17-1.69) and first ischemic stroke (odds ratio, 1.55; 95% confidence interval: 1.26-1.90). Results were consistent across various subgroups, including age, sex, baseline SBP, treatment compliance, and mean SBP, concomitant usage of calcium channel blocker during the first 12-month follow-up period. Similar trends were also found for coefficient of variation SBP, and SD or coefficient of variation DBP. However, there was no significant association between BP variability and first hemorrhagic stroke. CONCLUSION: In hypertensive adults with mild-to-moderate CKD, visit-to-visit variability in BP was significantly associated with the risk of subsequent first stroke.

Urinary Matrix Metalloproteinase-7 and Prediction of AKI Progression Post Cardiac Surgery.

AIMS: Early detection of patients at high risk for progressive acute kidney injury (AKI) after cardiac surgery remains a major challenge. We aim to evaluate the utility of urinary matrix metalloproteinase-7 (uMMP-7) and other reported biomarkers for predicting AKI progression during postoperative hospital stay. METHODS: We conducted a prospective, multicenter cohort study in 121 adult patients with stage 1 or 2 AKI after cardiac surgery. uMMP-7 and other well-reported biomarkers (uIL-18, uNGAL, and UACR) were measured at time of AKI clinical diagnosis. The primary outcome is the progression of AKI after cardiac surgery, defined as worsening of AKI stage (stage 1 to either stage 2 or stage 3 or from stage 2 to stage 3). RESULTS: A level of uMMP-7 > 7.8 µg/g Cr at time of AKI diagnosis conveyed an 8-fold risk of AKI progression as compared to those with uMMP-7 < 2.7 µg/g after adjusting for clinical risk factors. The performance of uMMP-7 for predicting progressive AKI was good with an AUC of 0.80. The combination of uMMP-7 and IL-18 produces the greatest AUC for predicting progressive AKI. Addition of uMMP-7 to the clinical risk factor model significantly improved risk reclassification for AKI progression. CONCLUSIONS: uMMP-7, measured at time of AKI clinical diagnosis, is a novel biomarker for predicting the progression of AKI after cardiac surgery. Adding uMMP-7 to the clinical risk factor model may be used as a noninvasive approach to identify a subpopulation that is at high risk for progressive AKI after cardiac surgery.

Urinary Angiotensinogen Predicts Renal Disease Activity in Lupus Nephritis.

Aims: A noninvasive indicator of renal histological lesions and disease activity in lupus nephritis (LN) is needed for timely and targeted treatment before overt renal injury. Here, we tested the utility of urinary angiotensinogen (UAGT) to predict renal disease activity in LN. Results: A prospective, three-stage study was performed in patients with LN. In stage I, UAGT was measured in 140 newly diagnosed LN patients. UAGT significantly increased in LN patients, correlating well with kidney angiotensinogen expression and histological activity. Patients with LN class IV exhibited the highest UAGT compared with other histopathological classes of LN. For identifying LN class IV, a particularly aggressive type of LN, UAGT outperformed the conventional clinical measures and improved their performance. In stage II, UAGT was monitored in 61 subjects from stage I for up to 12 months. UAGT decreased after induction therapy and remained low in patients with LN remission during follow-up. For predicting therapy success at month 12, the area under the receiver operating characteristics curve of UAGT reduction at month 4 was 0.83, outperforming that of 24-h proteinuria. In stage III, UAGT was monitored in 12 LN patients before, during, and after the onset of renal flares. An elevation in UAGT predicted recurrence of LN, and a decline in UAGT after a renal flare heralded the remission of disease before conventional clinical measures. Innovation and Conclusion: UAGT in LN is a promising indicator for dynamic surveillance of renal disease activity and prediction of renal flares. Antioxid. Redox Signal. 31, 1289-1301.

[Urinary soluble intercellular adhesion molecule-1 and vascular cellular adhesion molecule-1: potential biomarkers of active lupus nephritis].

OBJECTIVE: To determine the levels of urinary soluble intercellular adhesion molecule-1 (sICAM-1) and vascular cellular adhesion molecule-1 (VCAM-1) in patients with lupus nephritis (LN), and explore their correlation with renal disease activity. METHODS: Urine samples were collected from 92 renal biopsy-proven LN patients and 20 healthy controls. Renal disease activity was determined according to the ISN/RPS 2003 Revised Classification of Lupus Nephritis. The urine levels of sICAM-1 and VCAM-1 were detected by enzyme-linked immunosorbent assay, and the expressions of intrarenal ICAM-1 and VCAM-1 were evaluated by immunohistochemisty staining. RESULTS: Urinary sICAM-1 and VCAM-1 levels were elevated in LN patients compared with the controls. Significantly higher levels of urinary sICAM-1 and VCAM-1 were found in patients with active LN, who had also significantly increased intrarenal expressions of ICAM-1 and VCAM-1 compared with patients in remission. A strong positive correlation was noted between intrarenal expression and urine levels of ICAM-1 and VCAM-1. The receiver-operating characteristic (ROC) curve of urine sICAM-1 showed tan area under ROC curve of 0.874 for all participants in the test. A cutoff of 1095.00 pg/mg creatinine yielded a good sensitivity (0.945) and specificity (0.789). The ROC curve of urine VCAM-1 showed an area under ROC of 0.882 for all the participants, and a cutoff of 898.11 pg/mg creatinine yielded a good sensitivity (0.982) and specificity (0.667). CONCLUSION: Urine sICAM-1 and VCAM-1 levels are positively correlated with their intrarenal expressions and reflect the activity of the nephritis, and therefore they may serve as potential biomarkers for early diagnosis of active LN.