Analysis of cell-free fetal DNA in 16,843 pregnant women from a single center in China using targeted sequencing approach.

INTRODUCTION: Non-invasive prenatal testing (NIPT) using cell-free fetal DNA (cffDNA) has been widely accepted for detecting common fetal chromosome aneuploidies, but few large-scale studies have reported the kinetics of cffDNA concentration during gestation. This study examines cffDNA kinetics spanning gestational periods. METHODS: In this retrospective cohort study, cffDNA concentration from maternal plasma of 16,843 pregnant women between 4 and 39 weeks of pregnancy were determined by SNP-based targeted deep sequencing. RESULTS: Maternal plasma cffDNA could be detected as early as the fourth gestational week. After detection, cffDNA concentration begun to increase to the 39th week showing three conspicuous inflection points characterized by growth and stabilization phases. The rapid increase in cffDNA (∼1.19% per week) from the 4th to 9th week represents the first growth stage. The first plateau phase spanned from the 10th to 19th week (∼0.03% increase per week). cffDNA begun to rise dramatically (∼0.85% per week) from the 19th to 29th week, stabilizing at week 30 and onwards with only 0.27% increase per week representing the second plateau period. The proportion of cases with cffDNA ≥4% increased rapidly before the 10th gestational week with no significant increase from the 10th week onwards. About 92.00% of all the maternal plasma had a cffDNA greater than 4% from 10 weeks. DISCUSSION: We indicate that cffDNA had 3 inflection points at the 10th, 19th and 30th week of gestation, an observation not yet reported. Moreover, we show that cffDNA concentration has met the NIPT requirements after 9 weeks gestational age.

Urinary donor-derived cell-free DNA as a non-invasive biomarker for BK polyomavirus-associated nephropathy.

BK polyomavirus-associated nephropathy (BKPyVAN) is a common cause of allograft failure. However, differentiation between BKPyVAN and type I T cell-mediated rejection (TCMR) is challenging when simian virus 40 (SV40) staining is negative, because of the similarities in histopathology. This study investigated whether donor-derived cell-free DNA (ddcfDNA) can be used to differentiate BKPyVAN. Target region capture sequencing was applied to detect the ddcfDNAs of 12 recipients with stable graft function, 22 with type I TCMR, 21 with proven BKPyVAN, and 5 with possible PyVAN. We found that urinary ddcfDNA levels were upregulated in recipients with graft injury, whereas plasma ddcfDNA levels were comparable for all groups. The median urinary concentrations and fractions of ddcfDNA in proven BKPyVAN recipients were significantly higher than those in type I TCMR recipients (10.4 vs. 6.1 ng/mL, P<0.001 and 68.4% vs. 55.3%, P=0.013, respectively). Urinary ddcfDNA fractions (not concentrations) were higher in the BKPyVAN-pure subgroup than in the BKPyVAN-rejection-like subgroup (81.30% vs. 56.64%, P=0.025). With a cut-off value of 7.81 ng/mL, urinary ddcfDNA concentrations distinguished proven BKPyVAN from type I TCMR (area under the curve (AUC)=0.848, 95% confidence interval (95% CI): 0.734 to 0.963). These findings suggest that urinary ddcfDNA is a non-invasive biomarker which can reliably differentiate BKPyVAN from type I TCMR.

A novel urine cell-free DNA preservation solution and its application in kidney transplantation.

AIM: Urine cell-free DNA (cfDNA) is a new type of liquid biopsy biomarker used in tumours and allograft injury detection but is highly susceptible to degradation by the high nuclease activity of urine. This study presents a newly developed urine cfDNA preservation solution (AlloU), efficient for examining allograft injury in kidney transplant recipients (KTx). METHODS: We established urine-preserve solution called AlloU based on the response-surface methodology, with two commercial collection reagents (Streck and K2 EDTA preservation solution) included for analysis. A total of 120 urine samples from KTx patients, including morning, nocturnal and random urine from specific storage time were subjected to investigation. The urine total cfDNA concentration was quantified by fluorometry, fragment distribution was analysed by qPCR, and donor-derived cfDNA (ddcfDNA) was detected by next-generation sequencing. RESULTS: Urine total cfDNA concentration and fragment size of samples preserved with AlloU and Streck did not change significantly within 5 days whereas the ddcfDNA also did not change significantly within 7 days. However, compared with EDTA, the total cfDNA concentration increased significantly on the third day. When compare with different urine types, it was found that samples preserved with AlloU showed no significant differences in total cfDNA concentration, fragment size, and ddcfDNA concentration, however, the SD for morning urine was significantly smaller in total cfDNA and ddcfDNA concentration. CONCLUSION: To the best of our knowledge, this is the first report to verify the dynamics of urine cfDNA in KTx, especially in the analysis impact of different urine types on cfDNA detection.

The role of donor-derived cell-free DNA in the detection of renal allograft injury.

Donor-derived cell-free DNA refers to the cell-free DNA derived from apoptosis or necrosis of allograft tissue, circulating in the body fluids of patients after organ transplantation, and carries health information on the donor tissue. In the past two years, donor-derived cell-free DNA has rapidly become a research hotspot in the field of graft rejection detection after organ transplant. Recent published data have increased our understanding of donor-derived cell-free DNA in the field of kidney transplantation, especially in association with acute rejection. Donor-derived cell-free DNA is predicted to become the next-generation biomarker for the non-invasive detection of allograft rejection. This article reviews the research, involving donor-derived cell-free DNA in ischemia-reperfusion injury, delayed graft function, acute rejection (antibody mediated rejection and T-cell mediated rejection), and BK virus nephropathy. We further discuss the limitations of current research models and suggest directions for future study.

Nephrotoxicity Profile of Cadmium Revealed by Proteomics in Mouse Kidney.

Cadmium (Cd) is a highly toxic metal and kidney is its main target. However, the molecular effects and associated potential impacts of Cd-accumulated kidney have not been well investigated. In this study, mouse was used as a model to investigate the Cd-induced proteomic profile change in kidney, and a total of 34 differentially expressed proteins were detected by two-dimensional gel electrophoresis (2-DE) and further identified by matrix-assisted laser desorption/ionization time of flight mass spectrometry (MALDI-TOF-MS). Through Gene Ontology analysis and KEGG pathway annotation, it showed that Cd-regulated kidney metabolism and promoted renal damage and cell migration. By validation of Western blotting and RT-qPCR, metastasis-related proteins LIM and SH3 domain protein 1 (LASP1) and phosphoenolpyruvate carboxykinase/cytosolic [GTP] (PEPCK1) were confirmed to be upregulated; Acyl-CoA synthetase medium-chain family member 3 (ACSM3) was downregulated. Furthermore, carcinoma development-related proteins initiation factor 4A (eIF4A) and pyridoxine-5'-phosphate oxidase (PNPO) were upregulated, and pyridoxal kinase (PK) was downregulated. The downregulation of Na(+)/H(+) exchange regulatory cofactor (NHERF3) might promote renal damage which associated with decrease of transferrin (TRF) in kidney. Taken together, our results revealed proteomic profile of Cd-induced nephrotoxicity and provided data for further insights into the mechanisms of Cd toxicity.

Urine Donor-Derived Cell-Free DNA Helps Discriminate BK Polyomavirus-Associated Nephropathy in Kidney Transplant Recipients With BK Polyomavirus Infection.

Background: Studies have shown that plasma donor-derived cell-free DNA (dd-cfDNA) can predict renal allograft antibody-mediated rejection. This study was performed to evaluate the value of urine dd-cfDNA concentration and dd-cfDNA fraction (%) for discriminating BK polyomavirus-associated nephropathy (BKPyVAN) in kidney transplant recipients with urinary BK polyomavirus (BKPyV) infection. Methods: In this retrospective single-center observational study, we enrolled kidney transplant recipients who were diagnosed with urine BKPyV infection between August 2018 and May 2019 at the First Affiliated Hospital of Sun Yat-sen University. Urine dd-cfDNA was measured by using a novel target region capture sequencing methodology. The pathological diagnosis of BKPyVAN was confirmed by anti-SV40-T immunohistochemical staining and classified using the American Society for Transplantation schema. Receiver operating characteristic curve analysis was used to investigate the relations of urine dd-cfDNA and dd-cfDNA% to intrarenal allograft BKPyV infection states. Results: In total, 93 patients were enrolled, including 40 cases of proven BKPyVAN, seven cases of probable BKPyVAN, 23 cases of possible BKPyVAN, and 23 cases of resolving BKPyVAN. Urine dd-cfDNA level in proven BKPyVAN (22.09 ± 21.27 ng/ml) was comparable to that in probable BKPyVAN (15.64 ± 6.73 ng/ml, P = 0.434) but was significantly higher than that in possible BKPyVAN (5.60 ± 3.53 ng/ml) and resolving BKPyVAN (5.30 ± 3.34 ng/ml) (both Ps < 0.05). Urine dd-cfDNA% of proven BKPyVAN (0.71 ± 0.21) was lower than that of probable BKPyVAN (0.91 ± 0.04, P < 0.001), but was significantly higher than that of possible BKPyVAN (0.56 ± 0.30) and resolving BKPyVAN (0.46 ± 0.28) (both Ps < 0.05). For distinguishing biopsy-proven BKPyVAN from biopsy-excluded BKPyVAN, the discrimination capacity of urine dd-cfDNA (AUC: 0.842, 95% CI: 0.735, 0.918) was superior to that of plasma BKPyV DNA load (AUC: 0.660, 95% CI: 0.537, 0.769) with 0.181 (95% CI: 0.043, 0.319) difference between areas under ROC curves (P = 0.010). Conclusion: The elevated urine dd-cfDNA level may help discriminate BKPyVAN in kidney transplant recipients with BKPyV viruria.

A Noninvasive and Donor-independent Method Simultaneously Monitors Rejection and Infection in Patients With Organ Transplant.

BACKGROUND: Rejection and infection are 2 major complications affecting the health and survival of patients receiving an allograft organ transplantation. We describe a diagnostic assay that simultaneously monitors for rejection and infection in recipients of kidney transplant by sequencing of cell-free DNA (cfDNA) in plasma. METHODS: By using cfDNA in plasma, we established a noninvasive method that simultaneously monitors rejection and infection in patients with a history of organ transplant. A total of 6200 single-nucleotide polymorphisms were captured by liquid hybridization and sequenced by next-generation sequencing. The donor-derived cfDNA (ddcfDNA) level was calculated based on maximum likelihood estimation, without relying on the donor's genotype. We also analyzed the nonhuman cfDNA to test for infections in the patients' plasma. RESULTS: Artificial ddcfDNA levels quantified by a donor-dependent and donor-independent algorithm were significantly correlated, with the multivariate coefficient of determination, or R2 value, of 0.999. This technique was applied on 30 patients (32 samples) after kidney transplantation, and a significant difference was observed on the ddcfdNA levels between nonrejection and rejection. Furthermore, 1 BK virus infection and 1 cytomegalovirus infection were revealed by this method, and the enrichment efficiency of the viral sequences was 114 and 489 times, respectively, which are consistent with clinical results. CONCLUSION: This method can be used to simultaneously monitor for acute rejection as well as a broad spectrum of infections for patients of allograft organ transplant because it provides comprehensive information for clinicians to optimize immunosuppression therapy.

Molecular cloning, expression and characterization of secreted ferritin in the silkworm, Bombyx mori.

Ferritin is a ubiquitous iron storage protein which plays key role in regulating iron homeostasis and metabolism. In this paper, the ferritin heavy chain homologs (HCH) and light chain homologs (LCH) from Bombyx mori (BmFerHCH and BmFerLCH) were amplified through PCR and cloned into the expression vector pET-30a(+). The recombinant BmFerHCH and BmFerLCH expressed in Escherichia coli were in the form of insoluble inclusion bodies, indicating that the two proteins were not in their natural structural conformation. In order to obtain refolded ferritin in vitro, the inclusion bodies (BmFerHCH and/or BmFerLCH) were dissolved in denaturing buffer (100 mM Tris, 50 mM Glycine, 8 M urea, 5 mM DTT, pH 8.0) and then refolded in refolding buffer (100 mM Tris, 400 mM L-arginine, 0.2 mM PMSF, 0.5 mM DTT). The result showed that it was only when both BmFerHCH and BmFerLCH were present together in the denaturing buffer that refolding was successful and resulted in the formation of heteropolymers (H-L chain dimers) over homopolymers (H-H chain or L-L chain dimers). Moreover, the molecules (NaCl, Triton and glycerol) were found to enhance protein refolding. The optimum temperature, pH and ratios of BmFerHCH/BmFerLCH required for refolding were found to be 10 °C, pH 7, 1:1 or 1:2, respectively. Finally, the refolded ferritin had the ability to store iron, exhibited ferroxidase activity, and could withstand high temperatures and pH treatment, which is consistent with ferritin in other species.

Dynamics of early post-operative plasma ddcfDNA levels in kidney transplantation: a single-center pilot study.

Donor-derived cell-free DNA (ddcfDNA) is reported to be a promising noninvasive biomarker for acute rejection in organ transplant. However, studies on monitoring ddcfDNA dynamics during the early periods after organ transplantation are scarce. Our study assessed the dynamic variation in ddcfDNA in early period with various types and status of kidney transplantation. Target region capture sequencing used identifies ddcfDNA level in 21 kidney transplant recipients. Median ddcfDNA level was 20.69% at the initial time post-transplant, and decreased to 5.22% on the first day and stayed at the stable level after the second day. The ddcfDNA level in DCD (deceased donors) group (44.99%) was significantly higher than that in LDRT (living donor) group (10.24%) at initial time, P < 0.01. DdcfDNA level in DGF (delayed graft function) recipients was lower (23.96%) than that in non-DGF (47.74%) at the initial time, P = 0.89 (19.34% in DGF and 4.46% in non-DGF on the first day, P = 0.17). DdcfDNA level at initial time significantly correlated with serum creatinine (r2  = 0.219, P = 0.032) and warm ischemia time (r2  = 0.204, P = 0.040). Plasma ddcfDNA level decreased rapidly follow an L-shaped curve post-transplant, and level in DGF declined slower than non-DGF. The rebound of ddcfDNA level may indicate the occurrence of acute rejection.