Whole genome sequencing resolves 10 years diagnostic odyssey in familiar myxoma.

Carney complex (CNC) is an ultrarare disorder causing cutaneous and cardiac myxomas, primary pigmented nodular adrenocortical disease, hypophyseal adenoma, and gonadal tumours. Genetic alterations are often missed under routine genetic testing. Pathogenic variants in PRKAR1A are identified in most cases, while large exonic or chromosomal deletions have only been reported in a few cases. Our aim was to identify the causal genetic alteration in our kindred with a clinical diagnosis of CNC and prove its pathogenic role by functional investigation. Targeted testing of PRKAR1A gene, whole exome and whole genome sequencing (WGS) were performed in the proband, one clinically affected and one unaffected relative. WGS identified a novel, large, 10,662 bp (10.6 kbp; LRG_514t1:c.-10403_-7 + 265del; hg19, chr17:g.66498293_66508954del) deletion in the promoter of PRKAR1A in heterozygous form in the affected family members. The exact breakpoints and the increased enzyme activity in deletion carriers compared to wild type carrier were proved. Segregation analysis and functional evaluation of PKA activity confirmed the pathogenic role of this alteration. A novel deletion upstream of the PRKAR1A gene was proved to be the cause of CNC. Our study underlines the need for WGS in molecular genetic testing of patients with monogenic disorders where conventional genetic analysis fails.

Using a Chemical Biopsy for Graft Quality Assessment.

Kidney transplantation is a life-saving treatment for a large number of people with end-stage renal dysfunction worldwide. The procedure is associated with an increased survival rate and greater quality of patient's life when compared to conventional dialysis. Regrettably, transplantology suffers from a lack of reliable methods for organ quality assessment. Standard diagnostic techniques are limited to macroscopic appearance inspection or invasive tissue biopsy, which do not provide comprehensive information about the graft. The proposed protocol aims to introduce solid phase microextraction (SPME) as an ideal analytical method for comprehensive metabolomics and lipidomic analysis of all low molecular compounds present in kidneys allocated for transplantation. The small size of the SPME probe enables performance of a chemical biopsy, which enables extraction of metabolites directly from the organ without any tissue collection. The minimum invasiveness of the method permits execution of multiple analyses over time: directly after organ harvesting, during its preservation, and immediately after revascularization at the recipient's body. It is hypothesized that the combination of this novel sampling method with a high-resolution mass spectrometer will allow for discrimination of a set of characteristic compounds that could serve as biological markers of graft quality and indicators of possible development of organ dysfunction.

Normothermic Ex Vivo Liver Perfusion Prevents Intrahepatic Platelet Sequestration After Liver Transplantation.

BACKGROUND: The detrimental role of platelets in sinusoidal endothelial cell (SEC) injury during liver transplantation (LT) has been previously addressed after static cold storage (SCS), however, it is currently unknown after normothermic ex vivo liver perfusion (NEVLP). METHODS: Pig LT was performed with livers from heart-beating donors or donation after circulatory death (DCD) donors subjected to SCS or NEVLP (n = 5/group). RESULTS: All pigs except for 1 (DCD-SCS-group) survived 4 days. The heart-beating donor- and DCD-NEVLP-groups showed significantly lower aspartate transaminase-levels compared with the SCS-groups 3 hours post-LT (P = 0.006), on postoperative day (POD) 2 (P = 0.005), POD3 (P = 0.007), and on POD4 (P = 0.012). Post-LT total platelet count recovered faster in the NEVLP than in the SCS-groups at 12 hours (P = 0.023) and 24 hours (P = 0.0038). Intrahepatic sequestration of platelets was significantly higher in the SCS-groups 3 hours postreperfusion and correlated with severity of SEC injury. In both SCS-groups, levels of tumor growth factor-ß were higher 3 hours post-LT, on POD1 and on POD3. Moreover, platelet factor 4 levels and platelet-derived extracellular vesicles were increased in the SCS-groups. Hyaluronic acid levels were significantly higher in the SCS-groups, indicating a higher grade of endothelial cell dysfunction. Platelet inhibition achieved by pretreatment with clopidogrel (n = 3) partly reversed the detrimental effects on SEC injury and therefore provided further evidence of the important role of platelets in ischemia/reperfusion injury and SEC injury. CONCLUSIONS: Normothermic perfusion of liver grafts before transplantation effectively reduced platelet aggregation and SEC injury, which translated into an improved posttransplant organ function.

PPAR-gamma activation is associated with reduced liver ischemia-reperfusion injury and altered tissue-resident macrophages polarization in a mouse model.

BACKGROUND: PPAR-gamma (γ) is highly expressed in macrophages and its activation affects their polarization. The effect of PPAR-γ activation on Kupffer cells (KCs) and liver ischemia-reperfusion injury (IRI) has not yet been evaluated. We investigated the effect of PPAR-γ activation on KC-polarization and IRI. MATERIALS AND METHODS: Seventy percent (70%) liver ischemia was induced for 60mins. PPAR-γ-agonist or vehicle was administrated before reperfusion. PPAR-γ-antagonist was used to block PPAR-γ activation. Liver injury, necrosis, and apoptosis were assessed post-reperfusion. Flow-cytometry determined KC-phenotypes (pro-inflammatory Nitric Oxide +, anti-inflammatory CD206+ and anti-inflammatory IL-10+). RESULTS: Liver injury assessed by serum AST was significantly decreased in PPAR-γ-agonist versus control group at all time points post reperfusion (1hr: 3092±105 vs 4469±551; p = 0.042; 6hr: 7041±1160 vs 12193±1143; p = 0.015; 12hr: 5746±328 vs 8608±1259; p = 0.049). Furthermore, liver apoptosis measured by TUNEL-staining was significantly reduced in PPAR-γ-agonist versus control group post reperfusion (1hr:2.46±0.49 vs 6.90±0.85%;p = 0.001; 6hr:26.40±2.93 vs 50.13±8.29%; p = 0.048). H&E staining demonstrated less necrosis in PPAR-γ-agonist versus control group (24hr:26.66±4.78 vs 45.62±4.57%; p = 0.032). The percentage of pro-inflammatory NO+ KCs was significantly lower at all post reperfusion time points in the PPAR-γ-agonist versus control group (1hr:28.49±4.99 vs 53.54±9.15%; p = 0.040; 6hr:5.51±0.54 vs 31.12±9.58%; p = 0.009; 24hr:4.15±1.50 vs 17.10±4.77%; p = 0.043). In contrast, percentage of anti-inflammatory CD206+ KCs was significantly higher in PPAR-γ-agonist versus control group prior to IRI (8.62±0.96 vs 4.88 ±0.50%; p = 0.04). Administration of PPAR-γ-antagonist reversed the beneficial effects on AST, apoptosis, and pro-inflammatory NO+ KCs. CONCLUSION: PPAR-γ activation reduces IRI and decreases the pro-inflammatory NO+ Kupffer cells. PPAR-γ activation can become an important tool to improve outcomes in liver surgery through decreasing the pro-inflammatory phenotype of KCs and IRI.