Donor-Derived Cell-Free DNA for Kidney Allograft Surveillance after Conversion to Belatacept: Prospective Pilot Study.

Donor-derived cell-free DNA (dd-cfDNA) is used as a biomarker for detection of antibody-mediated rejection (ABMR) and other forms of graft injury. Another potential indication is guidance of immunosuppressive therapy when no therapeutic drug monitoring is available. In such situations, detection of patients with overt or subclinical graft injury is important to personalize immunosuppression. We prospectively measured dd-cfDNA in 22 kidney transplant recipients (KTR) over a period of 6 months after conversion to belatacept for clinical indication and assessed routine clinical parameters. Patient and graft survival was 100% after 6 months, and eGFR remained stable (28.7 vs. 31.1 mL/min/1.73 m2, p = 0.60). Out of 22 patients, 2 (9%) developed biopsy-proven rejection-one episode of low-grade TCMR IA and one episode of caABMR. While both episodes were detected by increase in creatinine, the caABMR episode led to increase in absolute dd-cfDNA (168 copies/mL) above the cut-off of 50 copies/mL, while the TCMR episode did show slightly increased relative dd-cfDNA (0.85%) despite normal absolute dd-cfDNA (22 copies/mL). Dd-cfDNA did not differ before and after conversion in a subgroup of 12 KTR with previous calcineurin inhibitor therapy and no rejection (12.5 vs. 25.3 copies/mL, p = 0.34). In this subgroup, 3/12 (25%) patients showed increase of absolute dd-cfDNA above the prespecified cut-off (50 copies/mL) despite improving eGFR. Increase in dd-cfDNA after conversion to belatacept is common and could point towards subclinical allograft injury. To detect subclinical TCMR changes without vascular lesions, additional biomarkers or urinary dd-cfDNA should complement plasma dd-cfDNA. Resolving CNI toxicity is unlikely to be detected by decreased dd-cfDNA levels. In summary, the sole determination of dd-cfDNA has limited utility in the guidance of patients after late conversion to belatacept. Further studies should focus on patients undergoing early conversion and include protocol biopsies at least for patients with increased dd-cfDNA.

Donor-Derived Cell-free DNA for Personalized Immunosuppression in Renal Transplantation.

BACKGROUND: The long-term outcomes of solid organ transplantation remain suboptimal. Therefore, appropriate biomarkers are needed in addition to immunosuppressive drugs and other traditional approaches for graft monitoring to achieve personalized immunosuppression and reduce premature graft loss. METHODS: Donor-derived cell-free DNA (dd-cfDNA) is a minimally invasive biomarker of cell death due to graft injury. It can be quantified using droplet digital polymerase chain reaction and next-generation sequencing. Fractional dd-cfDNA determination can be affected by changes in recipient cfDNA, such as those caused by leukopenia or infection, leading to false-positive or false-negative results, respectively. Absolute quantification of dd-cfDNA helps in overcoming this limitation. RESULTS: Overall, there is sufficient evidence of the clinical validity of dd-cfDNA. It detects rejection episodes early at an actionable stage and reflects the severity of graft injury without being rejection-specific. Owing to its high negative predictive value, dd-cfDNA is very useful for ruling out graft injury. Dd-cfDNA complements histological findings and can help in avoiding unnecessary biopsies. It indicates a response to rejection treatment and detects underimmunosuppression. CONCLUSIONS: Monitoring changes in dd-cfDNA over time may be helpful in adapting immunosuppression to prevent graft rejection. Moreover, serial dd-cfDNA determination may increase the effectiveness of transplant recipient surveillance and facilitate personalized immunosuppression when combined with other relevant clinical and diagnostic findings.

Donor-derived cell-free DNA as a diagnostic tool in transplantation.

There is a need to improve personalized immunosuppression in organ transplantation to reduce premature graft loss. Biomarkers are needed to better detect rejection, asymptomatic graft injury, and under-immunosuppression. Assessment of minimal necessary exposure to guide tapering and prevent immune activation is also important. There is robust clinical evidence from a large number of published studies supporting the role of dd-cfDNA for monitoring graft integrity and detection or exclusion of rejection. Dd-cfDNA indicates graft cell death without being rejection specific. It can be determined in plasma through droplet digital PCR using preselected SNPs or next generation sequencing. Changes in recipient cfDNA (e.g., by infection) can affect the results of dd-cfDNA fractional determination. This limitation can be overcome using absolute dd-cfDNA quantification. The combination of fractional and absolute determination including total cfDNA is recommended for meaningful interpretation of the results. The value proposition for the patient includes earlier transplant injury detection and intervention, less full blown rejection risk, an alternative to invasive biopsies, and personalized immunosuppression with potential for improved long-term outcome. Transplant physicians benefit from better immunosuppressive guidance and having an alternative when biopsies are refused or contraindicated. Further advantages are improved biopsy interpretation, less trial and error changes in immunosuppression, and less time dealing with complications. The laboratory medicine specialist can provide more effective services. Hospital management and insurance companies could benefit from more cost-effective surveillance of transplant recipients. Potential cost savings would result from fewer biopsies as a result of the tests' high negative predictive value, fewer re-transplantations, and less organ failure with return to dialysis. A pathway to implementation and metrics is suggested to measure the effectiveness of dd-cfDNA testing.

Molecular Characterization of the Response to Conventional Chemotherapeutics in Pro-B-ALL Cell Lines in Terms of Tumor Relapse.

Little is known about optimally applying chemotherapeutic agents in a specific temporal sequence to rapidly reduce the tumor load and to improve therapeutic efficacy. The clinical optimization of drug efficacy while reducing side effects is still restricted due to an incomplete understanding of the mode of action and related tumor relapse mechanisms on the molecular level. The molecular characterization of transcriptomic drug signatures can help to identify the affected pathways, downstream regulated genes and regulatory interactions related to tumor relapse in response to drug application. We tried to outline the dynamic regulatory reprogramming leading to tumor relapse in relapsed MLL-rearranged pro-B-cell acute lymphoblastic leukemia (B-ALL) cells in response to two first-line treatments: dexamethasone (Dexa) and cytarabine (AraC). We performed an integrative molecular analysis of whole transcriptome profiles of each treatment, specifically considering public knowledge of miRNA regulation via a network-based approach to unravel key driver genes and miRNAs that may control the relapse mechanisms accompanying each treatment. Our results gave hints to the crucial regulatory roles of genes leading to Dexa-resistance and related miRNAs linked to chemosensitivity. These genes and miRNAs should be further investigated in preclinical models to obtain more hints about relapse processes.

Elevated fractional donor-derived cell-free DNA during subclinical graft injury after liver transplantation.

Personalized immunosuppression (IS) promises to improve the balance of necessary control of alloreactivity and dose-dependent adverse effects of long-term IS such as kidney insufficiency, infections, and malignancies. The majority of liver transplantation (LT) recipients exhibit graft injuries (graft inflammation and/or fibrosis) that are not eligible for an IS reduction according to current Banff criteria, even when liver enzymes are normal or only marginally elevated. This cross-sectional study evaluated the noninvasive prediction of such subclinical graft injuries in surveillance liver biopsies via donor-derived cell-free DNA (dd-cfDNA). Absolute and fractional dd-cfDNA increased stepwise from patients without histological signs of rejection (n = 26) over subclinical graft injury (n = 61), including subclinical T cell-mediated rejection to clinical overt T cell-mediated rejection (n = 21). Thus, fractional plasma dd-cfDNA was significantly elevated paired to surveillance biopsies with relevant subclinical graft injury according to 2016 Banff criteria compared with those with minimal or absent histological graft injury. In contrast, the presence of donor-specific anti-human leukocyte antigen antibodies was not associated with the amount of dd-cfDNA. The sensitivity and specificity of fractional dd-cfDNA to noninvasively predict relevant subclinical graft injury was rather limited with 73% and 52% at the cutoff value of 2.1% fractional dd-cfDNA. The positive predictive value of fractional dd-cfDNA above 2.1% was 76% to noninvasively predict subclinical graft injury, calculated on the prevalence of graft injury in our prospective surveillance biopsy program, whereas the negative predictive values was not predictive (47%). In conclusion, dd-cfDNA has a rather limited diagnostic fidelity in addition to other noninvasive markers for the assessment of subclinical graft injury in personalized IS approaches after LT in a cross-sectional setting.

Temporary antimetabolite treatment hold boosts SARS-CoV-2 vaccination-specific humoral and cellular immunity in kidney transplant recipients.

Transplant recipients exhibit an impaired protective immunity after SARS-CoV-2 vaccination, potentially caused by mycophenolate (MPA) immunosuppression. Recent data from patients with autoimmune disorders suggest that temporary MPA hold might greatly improve booster vaccination outcomes. We applied a fourth dose of SARS-CoV-2 vaccine to 29 kidney transplant recipients during a temporary (5 weeks) MPA/azathioprine hold, who had not mounted a humoral immune response to previous vaccinations. Seroconversion until day 32 after vaccination was observed in 76% of patients, associated with acquisition of virus-neutralizing capacity. Interestingly, 21/25 (84%) calcineurin inhibitor-treated patients responded, but only 1/4 belatacept-treated patients responded. In line with humoral responses, counts and relative frequencies of spike receptor binding domain-specific (RBD-specific) B cells were markedly increased on day 7 after vaccination, with an increase in RBD-specific CD27++CD38+ plasmablasts. Whereas overall proportions of spike-reactive CD4+ T cells remained unaltered after the fourth dose, frequencies were positively correlated with specific IgG levels. Importantly, antigen-specific proliferating Ki67+ and in vivo-activated programmed cell death 1-positive T cells significantly increased after revaccination during MPA hold, whereas cytokine production and memory differentiation remained unaffected. In summary, antimetabolite hold augmented all arms of immunity during booster vaccination. These data suggest further studies of antimetabolite hold in kidney transplant recipients.

RNA-seq of nine canine prostate cancer cell lines reveals diverse therapeutic target signatures.

BACKGROUND: Canine prostate adenocarcinoma (PAC) and transitional cell carcinoma (TCC) are typically characterized by metastasis and chemoresistance. Cell lines are important model systems for developing new therapeutic strategies. However, as they adapt to culturing conditions and undergo clonal selection, they can diverge from the tissue from which they were originally derived. Therefore, a comprehensive characterization of cell lines and their original tissues is paramount. METHODS: This study compared the transcriptomes of nine canine cell lines derived from PAC, PAC metastasis and TCC to their respective original primary tumor or metastasis tissues. Special interests were laid on cell culture-related differences, epithelial to mesenchymal transition (EMT), the prostate and bladder cancer pathways, therapeutic targets in the PI3K-AKT signaling pathway and genes correlated with chemoresistance towards doxorubicin and carboplatin. RESULTS: Independent analyses for PAC, PAC metastasis and TCC revealed 1743, 3941 and 463 genes, respectively, differentially expressed in the cell lines relative to their original tissues (DEGs). While genes associated with tumor microenvironment were mostly downregulated in the cell lines, patient-specific EMT features were conserved. Furthermore, examination of the prostate and bladder cancer pathways revealed extensive concordance between cell lines and tissues. Interestingly, all cell lines preserved downstream PI3K-AKT signaling, but each featured a unique therapeutic target signature. Additionally, resistance towards doxorubicin was associated with G2/M cell cycle transition and cell membrane biosynthesis, while carboplatin resistance correlated with histone, m- and tRNA processing. CONCLUSION: Comparative whole-transcriptome profiling of cell lines and their original tissues identifies models with conserved therapeutic target expression. Moreover, it is useful for selecting suitable negative controls, i.e., cell lines lacking therapeutic target expression, increasing the transfer efficiency from in vitro to primary neoplasias for new therapeutic protocols. In summary, the dataset presented here constitutes a rich resource for canine prostate and bladder cancer research.

Graft-derived Cell-free DNA as a Noninvasive Biomarker of Cardiac Allograft Rejection: A Cohort Study on Clinical Validity and Confounding Factors.

BACKGROUND: Circulating graft-derived cell-free DNA (dd-cfDNA) is a new marker of cardiac allograft damage that is used for noninvasive rejection diagnostics. We performed dd-cfDNA (%) in heart transplant recipients during the first posttransplant year. METHODS: In 87 patients, serial dd-cfDNA determination at predefined time-points was performed in 770 single samples. dd-cfDNA fraction (%) was measured using an established universal droplet digital polymerase chain reaction method, providing same-day turn-around. Rejection was diagnosed according to clinical parameters and biopsies. RESULTS: Median dd-cfDNA (%) was high (5.36%) immediately after reperfusion and decreased to a median (interquartile range) of 0.10% (0.05%-0.24%) in clinically stable patients by postoperative day 10. Compared to dd-cfDNA (%) samples in clinically stable patients, values were higher (P < 0.001) in biopsy-proven rejection ISHLT 1R (0.42% [0.15%-0.53%]) and 2R rejection (0.84% [0.39%-0.97%]). Moreover, dd-cfDNA (%) was already significantly increased 9-30 days before biopsy-proven rejection (0.36% [0.20%-0.61%]). An as yet unknown finding was a slightly, but significantly (P < 0.0001) higher dd-cfDNA (%) value in samples of stable patients with pericardial effusions (PEs) (n = 94; 0.18% [0.07%-0.30%]) compared to samples of non-PE patients (n = 132; 0.07% [0.04%-0.17%]). Using a cutoff of 0.35%, sensitivity and specificity of dd-cfDNA for cardiac rejection were 0.76 and 0.83 (area under the curve [AUC] ROC-curve: 0.81 [95% confidence interval, 0.73-0.89]). Omitting PE samples from the control group yielded an AUC of 0.86 [95% confidence interval, 0.76-0.95]. Samples drawn <12 hours after endomyocardial biopsy showed high (0.40% [0.15%-1.21%]) dd-cfDNA values, also in ISHLT0R (0.36% [0.10%-0.60%]). CONCLUSIONS: dd-cfDNA plasma values were significantly associated with cardiac rejection. However, PE or improper sampling (eg, shortly after biopsy) should be considered as confounders for rejection diagnoses using dd-cfDNA.

An RNA-Seq-Based Framework for Characterizing Canine Prostate Cancer and Prioritizing Clinically Relevant Biomarker Candidate Genes.

Prostate cancer (PCa) in dogs is a highly malignant disease akin to its human counterpart. In contrast to the situation in humans, multi-gene approaches facilitating risk stratification of canine PCa are barely established. The aims of this study were the characterization of the transcriptional landscape of canine PCa and the identification of diagnostic, prognostic and/or therapeutic biomarkers through a multi-step screening approach. RNA-Sequencing of ten malignant tissues and fine-needle aspirations (FNA), and 14 nonmalignant tissues and FNAs was performed to find differentially expressed genes (DEGs) and deregulated pathways. The 4098 observed DEGs were involved in 49 pathways. These 49 pathways could be grouped into five superpathways summarizing the hallmarks of canine PCa: (i) inflammatory response and cytokines; (ii) regulation of the immune system and cell death; (iii) cell surface and PI3K signaling; (iv) cell cycle; and (v) phagosome and autophagy. Among the highly deregulated, moderately to strongly expressed DEGs that were members of one or more superpathways, 169 DEGs were listed in relevant databases and/or the literature and included members of the PCa pathway, oncogenes, prostate-specific genes, and druggable genes. These genes are novel and promising candidate diagnostic, prognostic and/or therapeutic canine PCa biomarkers.

Absolute or Relative Quantification of Donor-derived Cell-free DNA in Kidney Transplant Recipients: Case Series.

BACKGROUND: Donor-derived cell-free DNA (dd-cfDNA) is increasingly recognized as a valuable biomarker for acute transplant injury, with possible indications in the detection of cellular or humoral rejection and the guidance of immunosuppressive therapy. There is an ongoing debate on whether relative or absolute quantification of dd-cfDNA is more reliable for the detection of acute transplant injury. METHODS: We retrospectively reviewed all 22 kidney transplant recipients who underwent dd-cfDNA measurements (percentage and absolute) between April 2020 and April 2021 at our institution. Of these, 9 (41%) showed discrepancies between absolute (cutoff: 50 copies/mL) and relative (cutoff: 0.5%) quantification in at least 1 dd-cfDNA measurement. RESULTS: We report on 9 of 22 cases with discrepancies in relative and absolute quantification of dd-cfDNA, which were predominantly late posttransplant patients. We found bacterial and viral infections, as well as low leukocyte count from chronic myeloid leukaemia treatment, to be reasons for variability in total cell-free DNA (cfDNA), leading to inter- and intraindividual variability in relative dd-cfDNA quantification. When correlating dd-cfDNA quantification and biopsy results, as well as clinical course, our data indicate that relying solely on relative dd-cfDNA can lead to false-negative and false-positive results. CONCLUSIONS: In summary, these cases argue that absolute quantification of dd-cfDNA is better suited in patients with underlying conditions affecting total cfDNA levels and suggest using both absolute and relative dd-cfDNA together for higher reliability and interindividual comparability in the clinical setting. Especially for patients with chronic active antibody-mediated rejection, further studies on the use of dd-cfDNA are desirable.

Liquid biopsies: donor-derived cell-free DNA for the detection of kidney allograft injury.

In kidney transplantation, the use of minimally invasive damage biomarkers that are more sensitive and specific than plasma creatinine will be crucial to enable early, actionable detection or exclusion of structural kidney damage due to acute or chronic rejection. Donor-derived cell-free DNA (dd-cfDNA), which can be quantified, for example, through next-generation sequencing, droplet digital PCR and quantitative PCR, is a candidate biomarker with great potential for enabling comprehensive monitoring of allograft injury. dd-cfDNA has a favourable overall diagnostic performance for the detection of rejection and its high negative predictive value might be especially useful for avoiding unnecessary biopsies. Elevated dd-cfDNA levels have been shown to be detectable before graft injury can be clinically identified using current diagnostic methods. Moreover, dd-cfDNA falls rapidly to baseline levels after successful treatment for rejection owing to its short half-life. dd-cfDNA can detect graft injury caused by immune activation owing to insufficient immunosuppression and might therefore also help guide immunosuppression dosing. The fractional abundance of dd-cfDNA can be affected by changes in the recipient cfDNA (for example, due to infection or physical exercise) but the use of absolute quantification of dd-cfDNA overcomes this limitation. Serial dd-cfDNA determinations might therefore facilitate cost-effective personalized clinical management of kidney transplant recipients to reduce premature graft loss.

Cell-Free-DNA-Based Copy Number Index Score in Epithelial Ovarian Cancer-Impact for Diagnosis and Treatment Monitoring.

BACKGROUND: Chromosomal instability, a hallmark of cancer, results in changes in the copy number state. These deviant copy number states can be detected in the cell-free DNA (cfDNA) and provide a quantitative measure of the ctDNA levels by converting cfDNA next-generation sequencing results into a genome-wide copy number instability score (CNI-Score). Our aim was to determine the role of the CNI-Score in detecting epithelial ovarian cancer (EOC) and its role as a marker to monitor the response to treatment. METHODS: Blood samples were prospectively collected from 109 patients with high-grade EOC. cfDNA was extracted and analyzed using a clinical-grade assay designed to calculate a genome-wide CNI-Score from low-coverage sequencing data. Stored data from 241 apparently healthy controls were used as a reference set. RESULTS: Comparison of the CNI-Scores of primary EOC patients versus controls yielded sensitivities of 91% at a specificity of 95% to detect OC, respectively. Significantly elevated CNI-Scores were detected in primary (median: 87, IQR: 351) and recurrent (median: 346, IQR: 1891) blood samples. Substantially reduced CNI-Scores were detected after primary debulking surgery. Using a cut-off of 24, a diagnostic sensitivity of 87% for primary and recurrent EOC was determined at a specificity of 95%. CNI-Scores above this threshold were detected in 21/23 primary tumor (91%), 36/42 of platinum-eligible recurrent (85.7%), and 19/22 of non-platinum-eligible recurrent (86.3%) samples, respectively. CONCLUSION: ctDNA-quantification based on genomic instability determined by the CNI-Score was a biomarker with high diagnostic accuracy in high-grade EOC. The applied assay might be a promising tool for diagnostics and therapy monitoring, as it requires no a priori information about the tumor.

Suppression of the TGF-ß pathway by a macrolide antibiotic decreases fibrotic responses by ocular fibroblasts in vitro.

To elucidate and to inhibit post-surgical fibrotic processes after trabeculectomy in glaucoma therapy, we measured gene expression in a fibrotic cell culture model, based on transforming growth factor TGF-ß induction in primary human tenon fibroblasts (hTFs), and used Connectivity Map (CMap) data for drug repositioning. We found that specific molecular mechanisms behind fibrosis are the upregulation of actins, the downregulation of CD34, and the upregulation of inflammatory cytokines such as IL6, IL11 and BMP6. The macrolide antibiotic Josamycin (JM) reverses these molecular mechanisms according to data from the CMap, and we thus tested JM as an inhibitor of fibrosis. JM was first tested for its toxic effects on hTFs, where it showed no influence on cell viability, but inhibited hTF proliferation in a concentration-dependent manner. We then demonstrated that JM suppresses the synthesis of extracellular matrix (ECM) components. In hTFs stimulated with TGF-ß1, JM specifically inhibited α-smooth muslce actin expression, suggesting that it inhibits the transformation of fibroblasts into fibrotic myofibroblasts. In addition, a decrease of components of the ECM such as fibronectin, which is involved in in vivo scarring, was observed. We conclude that JM may be a promising candidate for the treatment of fibrosis after glaucoma filtration surgery or drainage device implantation in vivo.

Time-Dependent Apparent Increase in dd-cfDNA Percentage in Clinically Stable Patients Between One and Five Years Following Kidney Transplantation.

BACKGROUND: Donor-derived cell-free DNA (dd-cfDNA) is reportedly a valuable tool for graft surveillance following kidney transplantation (KTx). Possible changes in dd-cfDNA(%) reference values over time have not been evaluated. For long-term monitoring after KTx, changes in host cfDNA might represent a biasing factor in dd-cfDNA(%) determinations. METHODS: Plasma samples were obtained (n = 929) 12-60 months after engraftment in a cross-sectional cohort of 303 clinically stable KTx recipients. Total cfDNA(copies/mL), dd-cfDNA(%), and dd-cfDNA(copies/mL) were determined using droplet-digital PCR. Stability of threshold values in these stable KTx recipients over time was assessed by 80th, 85th, and 90th quantile regression. RESULTS: Upper percentiles of total cfDNA showed a significant decline of -1902, -3589, and -4753 cp/mL/log(month) (P = 0.014, <0.001, and 0.017, respectively), resulting in increasing dd-cfDNA(%) percentiles by 0.25, 0.46, and 0.72%/log(month) (P = 0.04, 0.001, and 0.002, respectively), with doubling of the 85th percentile value by 5 years. In contrast, dd-cfDNA(cp/mL) was stable during the observation period (P = 0.52, 0.29, and 0.39). In parallel increasing white blood cell counts and decreasing tacrolimus concentrations over time were observed. After 5 years, the median total cfDNA was still 1.6-fold (P < 0.001) higher in KTx recipients than in healthy controls (n = 135) and 1.4-fold (P < 0.001) higher than patients with other medical conditions (n = 364). CONCLUSIONS: The time-dependent decrease of host cfDNA resulted in an apparent increase of dd-cfDNA fraction in stable KTx patients. For long-term surveillance, measurement of absolute dd-cfDNA concentrations appears to be superior to percentages to minimize false positive results.

Donor-Derived Cell-Free DNA Testing in Solid Organ Transplantation: A Value Proposition.

BACKGROUND: There is a need to improve personalized immunosuppression in organ transplantation to reduce premature graft loss. More efficient biomarkers are needed to better detect rejection, asymptomatic graft injury, and under-immunosuppression. Assessment of minimal necessary exposure to guide tapering and to prevent immune activation is also important. Donor-derived cell-free DNA (dd-cfDNA) has become available for comprehensive monitoring of allograft integrity. A value proposition concept was applied to assess the potential benefits of dd-cfDNA to stakeholders (patient, transplant physician, laboratory medicine specialist, hospital management, insurance companies) involved in solid organ transplantation care. CONTENT: There is robust clinical evidence from more than 48 published studies supporting the role of dd-cfDNA for monitoring graft integrity and detection or exclusion of rejection. The value proposition framework was used to evaluate published key evidence regarding clinical validity, economic implications, and limitations of this approach. It has been shown that dd-cfDNA testing is essential for guiding earlier transplant injury intervention with potential for improved long-term outcome. SUMMARY: Monitoring dd-cfDNA offers a rapid and reproducible method to detect graft injuries at an early actionable stage without protocol biopsies and allows for more effective personalized immunosuppression. The appropriate use of dd-cfDNA testing can provide both clinical and economic benefits to all transplantation stakeholders.

Publisher Correction: Analysis of Copy-Number Variations and Feline Mammary Carcinoma Survival.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

Association of α/ß-Hydrolase D16B with Bovine Conception Rate and Sperm Plasma Membrane Lipid Composition.

We have identified a Holstein sire named Tarantino who had been approved for artificial insemination that is based on normal semen characteristics (i.e., morphology, thermoresistance, motility, sperm concentration), but had no progeny after 412 first inseminations, resulting in a non-return rate (NRdev) of -29. Using whole genome association analysis and next generation sequencing, an associated nonsense variant in the α/ß-hydrolase domain-containing 16B gene (ABHD16B) on bovine chromosome 13 was identified. The frequency of the mutant allele in the German Holstein population was determined to be 0.0018 in 222,645 investigated cattle specimens. The mutant allele was traced back to Whirlhill Kingpin (bornFeb. 13th, 1959) as potential founder. The expression of ABHD16B was detected by Western blotting and immunohistochemistry in testis and epididymis of control bulls. A lipidome comparison of the plasma membrane of fresh semen from carriers and controls showed significant differences in the concentration of phosphatidylcholine (PC), diacylglycerol (DAG), ceramide (Cer), sphingomyelin (SM), and phosphatidylcholine (-ether) (PC O-), indicating that ABHD16B plays a role in lipid biosynthesis. The altered lipid contents may explain the reduced fertilization ability of mutated sperms.

Analysis of Copy-Number Variations and Feline Mammary Carcinoma Survival.

Feline mammary carcinomas (FMCs) are highly malignant. As the disease-free survival (DFS) and overall survival (OS) are short, prognostication is crucial. Copy-number variations (CNVs) analysis by next-generation sequencing serves to identify critical cancer-related genomic regions. Thirty-three female cats with FMCs were followed during two years after surgery. Tumours represented tubulopapillary and solid carcinomas encompassing six molecular subtypes. Regardless of the histopathological diagnosis, molecular subtypes showed important differences in survival. Luminal A tumours exhibited the highest DFS (p = 0.002) and cancer-specific OS (p = 0.001), and the lowest amount of CNVs (p = 0.0001). In contrast, basal-like triple-negative FMCs had the worst outcome (DFS, p < 0.0001; and OS, p < 0.00001) and were the most aberrant (p = 0.05). In the multivariate analysis, copy-number losses (CNLs) in chromosome B1 (1-23 Mb) harbouring several tumour-repressors (e.g. CSMD1, MTUS1, MSR1, DBC2, and TUSC3) negatively influenced DFS. Whereas, copy-number gains (CNGs) in B4 (1-29 Mb) and F2 (64-82.3 Mb) comprising epithelial to mesenchymal transition genes and metastasis-promoting transcription factors (e.g. GATA3, VIM, ZEB1, and MYC) negatively influenced DFS and cancer-specific OS. These data evidence an association between specific CNVs in chromosomes B1, B4 and F2, and poor prognosis in FMCs.

Osteogenesis imperfecta in a male holstein calf associated with a possible oligogenic origin.

Background: Neuromusculoskeletal anomalies generally in combination with severe clinical symptoms, comprise a heterogeneous group of fairly common and mostly fatal disorders in man and animals. Osteogenesis imperfecta (OI), also known as brittle bone disease, causes bone fragility and deformity. Prominent extra-skeletal accessory manifestations of OI comprise blue/gray sclerae, hearing impairment, lung abnormalities and hypercalciuria. Cases of OI in cattle have been reported. However, no causative mutations have been identified in cattle so far.Aim: To report a possible oligogenic origin identified in a calf from clinically healthy parents suffering from OI.Materials and Methods: A neonatal embryo transfer male Holstein calf developing multiple fractures with bone tissue showing marked osteopenia was used for whole genome re-sequencing as well as its parents. In addition, 2,612 randomly chosen healthy Holstein cattle were genotyped as well as controls.Results: Sixteen candidate genes with potential protein-altering variants were selected revealing non-synonymous variants only within IFITM5 and CRTAP genes. However, in-depth gene analysis did not result in the identification of a single causative mutation in the OI calf.Conclusion: The analysis of the OI case revealed a possible oligogenic origin of the disease attributable to additive effects of three candidate genes, i.e., ABCA13, QRFPR, and IFTIM5.Clinical relevance: Most OI cases in humans and domestic animals reported so far are caused by distinct dominant or recessive monogenic mutations, therefore a potential oligogenic additive genetic effect is a novel finding. Furthermore, the case presented here demonstrates that cross-species genetic analyses might not always be straightforward.