Association between dd-cfDNA levels, de novo donor specific antibodies, and eGFR decline: An analysis of the DART cohort.

BACKGROUND: Donor-derived cell-free DNA (dd-cfDNA) is a marker of allograft injury in transplant recipients; however, the relationship between dd-cfDNA and other clinical parameters associated with adverse allograft outcomes is not well-characterized. METHODS: We performed a retrospective analysis of kidney transplant recipients from the DART cohort (ClinicalTrials.gov Identifier: NCT02424227) to evaluate the associations between eGFR decline, de novo donor-specific antibodies (dnDSA), and dd-cfDNA. RESULTS: Both elevated dd-cfDNA (≥1%) and dd-cfDNA variability (≥.34%) in the first post-transplant year were associated with decline in eGFR ≥25% in the second year (21.4% vs. 4.1%, P = .005; 25% vs. 3.6%, P = .002, respectively). Compared to samples from DSA negative patients, samples from patients with concurrent de novo HLA DSAs had higher dd-cfDNA levels (P < .0001). DISCUSSION: Abnormalities in dd-cfDNA levels are associated with clinical parameters commonly used as surrogate endpoints for adverse allograft outcomes, raising the possibility that molecular injury as characterized by dd-cfDNA could help identify patients at risk of these outcomes.

Biological function of unannotated transcription during the early development of Drosophila melanogaster.

Many animal and plant genomes are transcribed much more extensively than current annotations predict. However, the biological function of these unannotated transcribed regions is largely unknown. Approximately 7% and 23% of the detected transcribed nucleotides during D. melanogaster embryogenesis map to unannotated intergenic and intronic regions, respectively. Based on computational analysis of coordinated transcription, we conservatively estimate that 29% of all unannotated transcribed sequences function as missed or alternative exons of well-characterized protein-coding genes. We estimate that 15.6% of intergenic transcribed regions function as missed or alternative transcription start sites (TSS) used by 11.4% of the expressed protein-coding genes. Identification of P element mutations within or near newly identified 5' exons provides a strategy for mapping previously uncharacterized mutations to their respective genes. Collectively, these data indicate that at least 85% of the fly genome is transcribed and processed into mature transcripts representing at least 30% of the fly genome.

The transcriptional diversity of 25 Drosophila cell lines.

Drosophila melanogaster cell lines are important resources for cell biologists. Here, we catalog the expression of exons, genes, and unannotated transcriptional signals for 25 lines. Unannotated transcription is substantial (typically 19% of euchromatic signal). Conservatively, we identify 1405 novel transcribed regions; 684 of these appear to be new exons of neighboring, often distant, genes. Sixty-four percent of genes are expressed detectably in at least one line, but only 21% are detected in all lines. Each cell line expresses, on average, 5885 genes, including a common set of 3109. Expression levels vary over several orders of magnitude. Major signaling pathways are well represented: most differentiation pathways are "off" and survival/growth pathways "on." Roughly 50% of the genes expressed by each line are not part of the common set, and these show considerable individuality. Thirty-one percent are expressed at a higher level in at least one cell line than in any single developmental stage, suggesting that each line is enriched for genes characteristic of small sets of cells. Most remarkable is that imaginal disc-derived lines can generally be assigned, on the basis of expression, to small territories within developing discs. These mappings reveal unexpected stability of even fine-grained spatial determination. No two cell lines show identical transcription factor expression. We conclude that each line has retained features of an individual founder cell superimposed on a common "cell line" gene expression pattern.

Validation of a deep neural network-based algorithm supporting clinical management of adnexal mass.

Background: Conservative management of adnexal mass is warranted when there is imaging-based and clinical evidence of benign characteristics. Malignancy risk is, however, a concern due to the mortality rate of ovarian cancer. Malignancy occurs in 10-15% of adnexal masses that go to surgery, whereas the rate of malignancy is much lower in masses clinically characterized as benign or indeterminate. Additional diagnostic tests could assist conservative management of these patients. Here we report the clinical validation of OvaWatch, a multivariate index assay, with real-world evidence of performance that supports conservative management of adnexal masses. Methods: OvaWatch utilizes a previously characterized neural network-based algorithm combining serum biomarkers and clinical covariates and was used to examine malignancy risk in prospective and retrospective samples of patients with an adnexal mass. Retrospective data sets were assembled from previous studies using patients who had adnexal mass and were scheduled for surgery. The prospective study was a multi-center trial of women with adnexal mass as identified on clinical examination and indeterminate or asymptomatic by imaging. The performance to detect ovarian malignancy was evaluated at a previously validated score threshold. Results: In retrospective, low prevalence (N = 1,453, 1.5% malignancy rate) data from patients that received an independent physician assessment of benign, OvaWatch has a sensitivity of 81.8% [95% confidence interval (CI) 65.1-92.7] for identifying a histologically confirmed malignancy, and a negative predictive value (NPV) of 99.7%. OvaWatch identified 18/22 malignancies missed by physician assessment. A prospective data set had 501 patients where 106 patients with adnexal mass went for surgery. The prevalence was 2% (10 malignancies). The sensitivity of OvaWatch for malignancy was 40% (95% CI: 16.8-68.7%), and the specificity was 87% (95% CI: 83.7-89.7) when patients were included in the analysis who did not go to surgery and were evaluated as benign. The NPV remained 98.6% (95% CI: 97.0-99.4%). An independent analysis set with a high prevalence (45.8%) the NPV value was 87.8% (95% CI: 95% CI: 75.8-94.3%). Conclusion: OvaWatch demonstrated high NPV across diverse data sets and promises utility as an effective diagnostic test supporting management of suspected benign or indeterminate mass to safely decrease or delay unnecessary surgeries.

Through the Looking Glass: Unraveling the Stage-Shift of Acute Rejection in Renal Allografts.

Sub-optimal sensitivity and specificity in current allograft monitoring methodologies underscore the need for more accurate and reflexive immunosurveillance to uncover the flux in alloimmunity between allograft health and the onset and progression of rejection. QSant-a urine based multi-analyte diagnostic test-was developed to profile renal transplant health and prognosticate injury, risk of evolution, and resolution of acute rejection. Q-Score-the composite score, across measurements of DNA, protein and metabolic biomarkers in the QSant assay-enables this risk prognostication. The domain of immune quiescence-below a Q-Score threshold of 32-is well established, based on published AUC of 98% for QSant. However, the trajectory of rejection is variable, given that causality is multi-factorial. Injury and subtypes of rejection are captured by the progression of Q-Score. This publication explores the clinical utility of QSant across the alloimmunity gradient of 32-100 for the early diagnosis of allograft injury and rejection.

Differential analysis for high density tiling microarray data.

BACKGROUND: High density oligonucleotide tiling arrays are an effective and powerful platform for conducting unbiased genome-wide studies. The ab initio probe selection method employed in tiling arrays is unbiased, and thus ensures consistent sampling across coding and non-coding regions of the genome. These arrays are being increasingly used to study the associated processes of transcription, transcription factor binding, chromatin structure and their association. Studies of differential expression and/or regulation provide critical insight into the mechanics of transcription and regulation that occurs during the developmental program of a cell. The time-course experiment, which comprises an in-vivo system and the proposed analyses, is used to determine if annotated and un-annotated portions of genome manifest coordinated differential response to the induced developmental program. RESULTS: We have proposed a novel approach, based on a piece-wise function - to analyze genome-wide differential response. This enables segmentation of the response based on protein-coding and non-coding regions; for genes the methodology also partitions differential response with a 5' versus 3' versus intra-genic bias. CONCLUSION: The algorithm built upon the framework of Significance Analysis of Microarrays, uses a generalized logic to define regions/patterns of coordinated differential change. By not adhering to the gene-centric paradigm, discordant differential expression patterns between exons and introns have been identified at a FDR of less than 12 percent. A co-localization of differential binding between RNA Polymerase II and tetra-acetylated histone has been quantified at a p-value < 0.003; it is most significant at the 5' end of genes, at a p-value < 10-13. The prototype R code has been made available as supplementary material [see Additional file 1].

Rank-statistics based enrichment-site prediction algorithm developed for chromatin immunoprecipitation on chip experiments.

BACKGROUND: High density oligonucleotide tiling arrays are an effective and powerful platform for conducting unbiased genome-wide studies. The ab initio probe selection method employed in tiling arrays is unbiased, and thus ensures consistent sampling across coding and non-coding regions of the genome. Tiling arrays are increasingly used in chromatin immunoprecipitation (IP) experiments (ChIP on chip). ChIP on chip facilitates the generation of genome-wide maps of in-vivo interactions between DNA-associated proteins including transcription factors and DNA. Analysis of the hybridization of an immunoprecipitated sample to a tiling array facilitates the identification of ChIP-enriched segments of the genome. These enriched segments are putative targets of antibody assayable regulatory elements. The enrichment response is not ubiquitous across the genome. Typically 5 to 10% of tiled probes manifest some significant enrichment. Depending upon the factor being studied, this response can drop to less than 1%. The detection and assessment of significance for interactions that emanate from non-canonical and/or un-annotated regions of the genome is especially challenging. This is the motivation behind the proposed algorithm. RESULTS: We have proposed a novel rank and replicate statistics-based methodology for identifying and ascribing statistical confidence to regions of ChIP-enrichment. The algorithm is optimized for identification of sites that manifest low levels of enrichment but are true positives, as validated by alternative biochemical experiments. Although the method is described here in the context of ChIP on chip experiments, it can be generalized to any treatment-control experimental design. The results of the algorithm show a high degree of concordance with independent biochemical validation methods. The sensitivity and specificity of the algorithm have been characterized via quantitative PCR and independent computational approaches. CONCLUSION: The algorithm ranks all enrichment sites based on their intra-replicate ranks and inter-replicate rank consistency. Following the ranking, the method allows segmentation of sites based on a meta p-value, a composite array signal enrichment criterion, or a composite of these two measures. The sensitivities obtained subsequent to the segmentation of data using a meta p-value of 10-5, an array signal enrichment of 0.2 and a composite of these two values are 88%, 87% and 95%, respectively.

Extensive sequencing of seven human genomes to characterize benchmark reference materials.

The Genome in a Bottle Consortium, hosted by the National Institute of Standards and Technology (NIST) is creating reference materials and data for human genome sequencing, as well as methods for genome comparison and benchmarking. Here, we describe a large, diverse set of sequencing data for seven human genomes; five are current or candidate NIST Reference Materials. The pilot genome, NA12878, has been released as NIST RM 8398. We also describe data from two Personal Genome Project trios, one of Ashkenazim Jewish ancestry and one of Chinese ancestry. The data come from 12 technologies: BioNano Genomics, Complete Genomics paired-end and LFR, Ion Proton exome, Oxford Nanopore, Pacific Biosciences, SOLiD, 10X Genomics GemCode WGS, and Illumina exome and WGS paired-end, mate-pair, and synthetic long reads. Cell lines, DNA, and data from these individuals are publicly available. Therefore, we expect these data to be useful for revealing novel information about the human genome and improving sequencing technologies, SNP, indel, and structural variant calling, and de novo assembly.

T2 relaxation time measurements in osteoarthritis.

Quantification of changes in T(2) relaxation time, in human cartilage, with progression of osteoarthritis (OA), and evaluation of qualitative correlations with clinical evaluation, histology and polarized light microscopy (PLM). Cartilage-bone plugs were harvested from fresh cadaveric knees (n = 10) and specimens after surgical knee replacement (n = 2) at 12 locations, including lateral and medial sides of tibia, femora and patella. Magnetic resonance imaging was performed at 1.5 Tesla using a.2D spin echo sequence. Histological slices were assessed for OA severity through a grading scale based on combined histological and PLM results. T(2) values in clinically moderate OA were generally higher than in severe OA and normal cartilage. Significant association was established between normal and early OA subjects and T(2) variation, in the medial compartment of the knee (p < 0.05) but especially in the medial tibial cartilage (p < 0.00005). As expected, medial and lateral tibio-femoral compartments underwent more severe degeneration. Additionally, there were intracompartmental variation of the relaxation times and histological patterns, which demonstrate the underlying focal involvement of OA in the knee. Furthermore, T(2) values reflected OA pathogenesis with a positive correlation with histology grading scale. Finally, increased T(2) is correlated to histological degeneration of cartilage and may be a good marker for early OA in tibial articular cartilage.

RNA maps reveal new RNA classes and a possible function for pervasive transcription.

Significant fractions of eukaryotic genomes give rise to RNA, much of which is unannotated and has reduced protein-coding potential. The genomic origins and the associations of human nuclear and cytosolic polyadenylated RNAs longer than 200 nucleotides (nt) and whole-cell RNAs less than 200 nt were investigated in this genome-wide study. Subcellular addresses for nucleotides present in detected RNAs were assigned, and their potential processing into short RNAs was investigated. Taken together, these observations suggest a novel role for some unannotated RNAs as primary transcripts for the production of short RNAs. Three potentially functional classes of RNAs have been identified, two of which are syntenically conserved and correlate with the expression state of protein-coding genes. These data support a highly interleaved organization of the human transcriptome.

Temporal profile of replication of human chromosomes.

Chromosomes in human cancer cells are expected to initiate replication from predictably localized origins, firing reproducibly at discrete times in S phase. Replication products obtained from HeLa cells at different stages of S phase were hybridized to cDNA and genome tiling oligonucleotide microarrays to determine the temporal profile of replication of human chromosomes on a genome-wide scale. About 1,000 genes and chromosomal segments were identified as sites containing efficient origins that fire reproducibly. Early replication was correlated with high gene density. An acute transition of gene density from early to late replicating areas suggests that discrete chromatin states dictate early versus late replication. Surprisingly, at least 60% of the interrogated chromosomal segments replicate equally in all quarters of S phase, suggesting that large stretches of chromosomes are replicated by inefficient, variably located and asynchronous origins and forks, producing a pan-S phase pattern of replication. Thus, at least for aneuploid cancer cells, a typical discrete time of replication in S phase is not seen for large segments of the chromosomes.

Transcriptional maps of 10 human chromosomes at 5-nucleotide resolution.

Sites of transcription of polyadenylated and nonpolyadenylated RNAs for 10 human chromosomes were mapped at 5-base pair resolution in eight cell lines. Unannotated, nonpolyadenylated transcripts comprise the major proportion of the transcriptional output of the human genome. Of all transcribed sequences, 19.4, 43.7, and 36.9% were observed to be polyadenylated, nonpolyadenylated, and bimorphic, respectively. Half of all transcribed sequences are found only in the nucleus and for the most part are unannotated. Overall, the transcribed portions of the human genome are predominantly composed of interlaced networks of both poly A+ and poly A- annotated transcripts and unannotated transcripts of unknown function. This organization has important implications for interpreting genotype-phenotype associations, regulation of gene expression, and the definition of a gene.

Osteoarthritis: MR imaging findings in different stages of disease and correlation with clinical findings.

PURPOSE: To determine whether knee pain, stiffness, and limited function in patients with different stages of osteoarthritis correlate with the degree of disease assessed on magnetic resonance (MR) images and radiographs. MATERIALS AND METHODS: Radiographs in 50 patients with varying degrees of osteoarthritis of the knee were assessed by using the the Western Ontario and McMaster University (WOMAC) osteoarthritis index and the Kellgren-Lawrence (KL) scale. MR images were obtained and analyzed by two readers for cartilage lesions, bone marrow edema pattern, and ligamentous and meniscal lesions. RESULTS: Thirteen of 16 knees with a KL score of 4 showed full-thickness cartilage lesions and bone marrow edema pattern. Cruciate ligament tears were found in five of 12 knees with a KL score of 3 and in nine of 16 knees with a KL score of 4. While the KL score correlated significantly (P <.05) with the grade of cartilage lesions, and a substantially higher percentage of lesions with higher KL scores were found on MR images, the correlations between MR imaging findings and KL score versus clinical findings were not significant (P >.05). Significant differences between WOMAC scores were found only for the grades of cartilage lesions (P <.05). CONCLUSION: Cartilage lesions, bone marrow edema pattern, and meniscal and ligamentous lesions were frequently demonstrated on MR images in patients with advanced osteoarthritis. Clinical findings showed no significant correlations with KL score and extent of findings at MR imaging.

Magnetic resonance imaging of normal and osteoarthritic trabecular bone structure in the human knee.

OBJECTIVE: To use high-resolution magnetic resonance imaging (MRI) to evaluate the trabecular bone structure in the distal femur and the proximal tibia and its to correlate the findings with different stages of osteoarthritis (OA) of the human knee. METHODS: Axial images of the distal femur and proximal tibia were obtained at 1.5 T in patients without and with mild OA and with severe OA. The spatial resolution was 195 x 195 microm(2) with a 1-mm slice thickness. Apparent measures of trabecular bone volume fraction (BV/TV), trabecular number (Tb.N), trabecular separation (Tb.Sp), and trabecular thickness (Tb.Th) were calculated. RESULTS: Significant differences existed in the trabecular bone structure of the femur and tibia. Differences in trabecular bone structure between the tibia and the femur decreased with the degree of OA. The apparent BV/TV, Tb.N, and Tb.Sp in the femoral condyles could be used to differentiate healthy patients or patients with mild OA from patients with severe OA (P < 0.05). Among individuals, the structural variation of the lateral and medial femoral condyle was indicative of the extent of the disease. CONCLUSION: High-resolution MRI of the knee joint can provide a noninvasive assessment of trabecular bone structure. Trabecular bone structure, determined by high-resolution MRI, shows significant variation in patients with varying degrees of OA. The impact of OA on trabecular bone is different in the tibia than in the femur, and this difference depends on the extent of the disease.