Noninvasive Detection of Urothelial Carcinoma by Cost-effective Low-coverage Whole-genome Sequencing from Urine-Exfoliated Cell DNA.

PURPOSE: Urothelial carcinoma is a malignant cancer with frequent chromosomal aberrations. Here, we investigated the application of a cost-effective, low-coverage whole-genome sequencing technology in detecting all chromosomal aberrations. EXPERIMENTAL DESIGN: Patients with urothelial carcinomas and nontumor controls were prospectively recruited in clinical trial NCT03998371. Urine-exfoliated cell DNA was analyzed by Illumina HiSeq XTen, followed by genotyping with a customized bioinformatics workflow named Urine Exfoliated Cells Copy Number Aberration Detector (UroCAD). RESULTS: In the discovery phase, urine samples from 126 patients with urothelial carcinomas and 64 nontumor disease samples were analyzed. Frequent chromosome copy-number changes were found in patients with tumor as compared with nontumor controls. A novel diagnosis model, UroCAD, was built by incorporating all the autosomal chromosomal changes. The model reached performance of AUC = 0.92 (95% confidence interval, 89.4%-97.3%). At the optimal cutoff, |Z| ≥ 3.21, the sensitivity, specificity, and accuracy were 82.5%, 96.9%, and 89.0%, respectively. The prediction positivity was found correlated with tumor grade (P = 0.01). In the external validation cohort of 95 participants, the UroCAD assay identified urothelial carcinomas with an overall sensitivity of 80.4%, specificity of 94.9%, and AUC of 0.91. Meanwhile, UroCAD assay outperformed cytology tests with significantly improved sensitivity (80.4% vs. 33.9%; P < 0.001) and comparable specificity (94.9% vs. 100%; P = 0.49). CONCLUSIONS: UroCAD could be a robust urothelial carcinoma diagnostic method with improved sensitivity and similar specificity as compared with cytology tests. It may be used as a noninvasive approach for diagnosis and recurrence surveillance in urothelial carcinoma prior to the use of cystoscopy, which would largely reduce the burden on patients.

Detection of four polymorphisms in 5' upstream region of PNPLA2 gene and their associations with economic traits in pigs.

As an important triglyceride hydrolase in mammalian cells, patatin-like phospholipase domain-containing 2 (PNPLA2) predominantly performs the first step in triglyceride hydrolysis. The objective of this study was to detect and evaluate the effects of mutations in the 5' upstream region of porcine PNPLA2 gene with fat deposition and carcass traits. Four single nuclear polymorphisms were identified, including g.161969 T>C, g.161962 A>G, g.161953 C>G and g.161904 G>T, and subsequently genotyped in five pure breeds. Three haplotypes were constructed, including H1(CGGT), H2(TACG) and H3(CACT), which were the most abundant haplotypes in Duroc (0.75), Landrace (0.78) and Chinese indigenous breeds (>0.73), respectively. Duroc individuals with the H1H1 diplotype always exhibited the lowest feed conversion ratio (FCR) (P < 0.05), while H2H2 had the thickest backfat thickness (P < 0.05). Landrace individuals with H2H3 had lower backfat thickness (P < 0.05), higher muscle thickness (P < 0.05) and estimated lean meat percentage (P < 0.05) than those with diplotype H2H2 and H3H3. Luciferase assay indicated pGL3-basic-H2 had the highest activity and pGL3-basic-H1 had the lowest activity in driving reporter gene transcription in HEK293 cells in vitro. In H1 haplotype, two GR binding sites and an ERα binding site were predicted to be introduced. While in H2 and H3, there were other transcriptional factor binding sites predicted in H2 and H3, such as Sp1, AP-2 and CAC-binding proteins, which were broadly expressed transcription factors and capable of contributing to basal promoter activity. The reduced basal promoter activity of H1 may be due to the lack of inducement for GR and ERα binding sites in HEK293 cells. The identified functional polymorphisms provide new evidence of PNPLA2 as an important candidate gene for fat deposition and carcass traits in pigs.