Deep phenotyping: symptom annotation made simple with SAMS.

Precision medicine needs precise phenotypes. The Human Phenotype Ontology (HPO) uses clinical signs instead of diagnoses and has become the standard annotation for patients\u27 phenotypes when describing single gene disorders. Use of the HPO beyond human genetics is however still limited. With SAMS (Symptom Annotation Made Simple), we want to bring sign-based phenotyping to routine clinical care, to hospital patients as well as to outpatients. Our web-based application provides access to three widely used annotation systems: HPO, OMIM, Orphanet. Whilst data can be stored in our database, phenotypes can also be imported and exported as Global Alliance for Genomics and Health (GA4GH) Phenopackets without using the database. The web interface can easily be integrated into local databases, e.g. clinical information systems. SAMS offers users to share their data with others, empowering patients to record their own signs and symptoms (or those of their children) and thus provide their doctors with additional information. We think that our approach will lead to better characterised patients which is not only helpful for finding disease mutations but also to better understand the pathophysiology of diseases and to recruit patients for studies and clinical trials. SAMS is freely available at https://www.genecascade.org/SAMS/

Serological Response to Three, Four and Five Doses of SARS-CoV-2 Vaccine in Kidney Transplant Recipients

Mortality from COVID-19 among kidney transplant recipients (KTR) is high, and their response to three vaccinations against SARS-CoV-2 is strongly impaired. We retrospectively analyzed the serological response of up to five doses of the SARS-CoV-2 vaccine in KTR from 27 December 2020 until 31 December 2021. Particularly, the influence of the different dose adjustment regimens for mycophenolic acid (MPA) on serological response to fourth vaccination was analyzed. In total, 4277 vaccinations against SARS-CoV-2 in 1478 patients were analyzed. Serological response was 19.5% after 1203 basic immunizations, and increased to 29.4%, 55.6%, and 57.5% in response to 603 third, 250 fourth, and 40 fifth vaccinations, resulting in a cumulative response rate of 88.7%. In patients with calcineurin inhibitor and MPA maintenance immunosuppression, pausing MPA and adding 5 mg prednisolone equivalent before the fourth vaccination increased the serological response rate to 75% in comparison to the no dose adjustment (52%) or dose reduction (46%). Belatacept-treated patients had a response rate of 8.7% (4/46) after three vaccinations and 12.5% (3/25) after four vaccinations. Except for belatacept-treated patients, repeated SARS-CoV-2 vaccination of up to five times effectively induces serological response in kidney transplant recipients. It can be enhanced by pausing MPA at the time of vaccination

The influence of the SNP rs2075820 on the development and the course of gastric cancer and on the function of the NOD1-receptor

Die chronische Infektion mit Helicobacter pylori ist der stärkste Risikofaktor für das Magenkarzinom. Die Erkennung des Bakteriums erfolgt über den intrazellulär lokalisierten, mustererkennenden Rezeptor NOD1. Dieser vermittelt über die Aktivierung des Transkriptionsfaktors NF-ĸB eine Zytokinantwort, unter anderem mit Ausschüttung von IL-1β. Der SNP rs2075820 kodiert für einen Basenaustausch im NOD1-Gen. Er führt zu einem Aminosäureaustausch in der NBD-Domäne des Rezeptors. Ziel dieser retrospektiven monozentrischen Studie war es, die epidemiologische Bedeutung der Allele des SNPs rs2075820 für das Magenkarzinom zu untersuchen. In einer Überexpressionsstudie in HEK293-Zellen und in Stimulationsexperimenten an humanen Monozyten sollte die Auswirkung der rs2075820-Genotypen auf die Funktion des NOD1-Rezeptors studiert werden. 269 Magenkarzinompatienten und 188 gesunde Kontrollprobanden wurden mittels LightCycler®-PCR für den SNP rs2075820 genotypisiert und die Allel- und Genotypfrequenzen miteinander verglichen. Im Patientenkollektiv wiesen 54 % den G/G-, 38 % den G/A- und 8 % den A/A-Genotyp auf. Dies entsprach der Genotypenverteilung im Kontrollkollektiv. Bei den Patienten mit dem A/A-Genotyp wurde das Magenkarzinom in einem signifikant früheren Tumorstadium diagnostiziert als beim G/A- und beim G/G-Genotyp; die Hälfte der Träger des A/A-Genotyps wurden im UICC-Stadium I diagnostiziert, die Träger des G/A- und G/G-Genotyps nur in jeweils einem Viertel der Fälle. Patienten mit dem A/A-Genotyp wiesen zudem seltener Fernmetastasen bei Erstdiagnose auf und wurden häufiger mit primär kurativer Intention behandelt. Sie überlebten deutlich länger als solche mit den Genotypen G/A bzw. G/G. Zur funktionellen Charakterisierung des SNPs rs2075820 wurden beide Allele in HEK293-Zellen überexprimiert, eine Stimulation mit dem NOD1-spezifischen Liganden iE-DAP durchgeführt und anschließend in einem Reportergen-Assay die NF-ĸB-Aktivierung gemessen. Das A-Allel wies eine stark verminderte Liganden-vermittelte Aktivierung des Transkriptionsfaktors NF-ĸB auf. Eine Sequenzierung der eingesetzten Vektoren zeigte jedoch, dass der NOD1-Referenz Vektor, der in der Mutagenese eingesetzt wurde, an Position 1733 eine Base enthält, die von der NOD1-Referenzsequenz abweicht und zu einem Aminosäureaustausch führt. Bei den Stimulationsexperimenten an humanen Monozyten wiesen Monozyten mit dem A/A-Genotyp im Gegensatz zu Monozyten mt dem G/G-Genotyp eine abgeschwächte ligandenvermittelte IL-1β-Ausschüttung auf. Die Genotypisierungsstudie zeigte, dass weder einzelne Allele noch Genotypen des SNPs rs2075820 mit dem Magenkarzinom assoziiert sind. Jedoch erwies sich der A/A-Genotyp durch ein lokal begrenztes Tumorstadium bei Diagnosestellung und ein längeres medianes Überleben als ein prognostisch günstiger Faktor. Die funktionellen Untersuchungen weisen dabei auf eine verringerte Aktivierbarkeit des NOD1-Rezeptors bei dem A/A-Genotyp hin. Die Aussagekraft des Überexpressionsversuchs in HEK-Zellen wird hauptsächlich durch den Basenaustasch im Referenz-Vektor limitiert, die der Monozytenstimulatiosexperimente vor allem durch die Vielzahl an experimentellen Schritten und die daraus resultierenden möglichen Fehlerquellen. Weitere Experimente sind notwendig, um die Auswirkung des SNP rs2075820 auf die Funktion des NOD1-Rezeptors genauer und umfassend beschreiben zu können.Chronic infection with H. pylori is the strongest risk factor for gastric cancer. The intracellular pattern recognition receptor NOD1 activates a cytokine response via the transcription factor NF-ĸB in response to H. pylori infection. Among others, the cytokine IL-1β is released. Aim of this retrospective single centre study was to examine the epidemiologic relevance of the non-synonymous SNP rs2075820, which leads to a glutamate (genotype G) to lysine (genotype A) substitution in the nucleotide binding domain, for gastric cancer. In an overexpression experiment in HEK293 cells and by stimulation of humane monocytes, we studied the effect of the different genotypes on the function of the NOD1 receptor. We genotyped 269 patients with gastric cancer and 188 healthy controls by quantitative PCR and compared the allele and genotype frequencies between the two groups. The genotype frequencies in patients with gastric cancer were 54 % (G/G), 38 % (G/A), and 8 % (A/A). This corresponded to the genotype frequencies determined in our healthy controls. Patient with the A/A genotype were significantly more often diagnosed at an early tumour stage compared to the G/A and G/G genotype; about 50% of the A/A genotype carriers were diagnosed in the UICC tumour stage I compared to only 25 % of the patients carrying the G/A or G/G genotype. Patients with the A/A genotype less often presented metastatic disease at the time of diagnosis and were more often treated with curative intention. Their mean survival time after diagnosis was longer than that of patients with G/G or G/A genotypes. To functionally characterise the SNP rs2075820, we overexpressed both alleles in HEK296 cells and later on stimulated these cells with the NOD1-specific ligand ieDAP. Finally, the expression of NF-ĸB was measured in a reporter gene assay. Cells expressing the A allele displayed a markedly diminished activity of NF-ĸB. In the monocyte stimulation experiment, human monocytes with the A/A genotype showed diminished ligand-induced IL-1β secretion compared to those with the G/G genotype. The genotype study showed no association between the SNP rs2075820 and the incidence of gastric cancer. However the A/A genotype was associated with a favourable prognosis with a longer median survival time and an earlier tumour stage at the time of diagnosis. The overexpression experiments indicate that the NOD1 receptor encoded by the A allele may be less activatable. However the validity of the overexpression experiments is limited , because complete sequencing of the vectors for both alleles revealed that both carry a synonymous mutation from the reference sequence which results in a leucin to proline substitution in the NOD1 protein. The monocyte simulation assay has a complex set up and is hence prone to errors. For a more precise characterisation of the effects of the rs2075820 alleles on the function of the NOD1 receptor, further studies are needed.

Urinary NGAL-Positive Acute Kidney Injury and Poor Long-term Outcomes in Hospitalized Patients

Neutrophil gelatinase−associated lipocalin (NGAL) is a widely studied biomarker of renal tubular injury. Urinary NGAL (uNGAL) during acute kidney injury (AKI) predicts short-term adverse outcomes. However, the long-term predictive value is unknown. Methods: We performed a prospective observational study of 145 patients with hospital-acquired AKI according to Risk, Injury, Failure, Loss of kidney function, and End-stage kidney disease (RIFLE) criteria and analyzed the long-term predictive value of uNGAL at the time of AKI. We defined a composite outcome of all-cause mortality and the development of end-stage renal disease (ESRD). Results: In all, 61 AKI patients died and 22 developed ESRD within 6 months. The uNGAL levels were significantly higher in patients with poor long-term outcomes. uNGAL levels ≥362 μg/l (highest quartile) and uNGAL levels between 95 and 362 μg/l (third quartile) were associated with hazard ratios of 3.7 (95% confidence interval, 2.1–6.5) and 1.9 (1.1–3.5), respectively, compared with uNGAL levels <95 μg/l (lower quartiles). After 6 months, 67% and 43% of patients within the highest and third uNGAL quartile, respectively, had either progressed to ESRD or died, compared to only 21% of patients with uNGAL in the lower 2 quartiles (P < 0.001). In multivariable Cox regression analyses accounting for conventional predictors, uNGAL was the strongest independent predictor of adverse long-term outcomes. The association of uNGAL levels and poor long-term outcomes remained significant in the subgroup of 107 AKI survivors discharged without requiring dialysis (P = 0.002). Discussion: These data indicate that elevated uNGAL levels at AKI diagnosis predict poor long-term outcomes

Targeting Cholesterol Metabolism as Efficient Antiviral Strategy Against the Hepatitis E Virus

BACKGROUND AND AIMS: The Hepatitis E virus hijacks the endosomal system for its release. These structures are highly dependent on cholesterol. Hence, this study investigates the impact of HEV on cholesterol-metabolism, the effect of intracellular cholesterol content on HEV-release and the potential of cholesterol-modulators to serve as antivirals. METHODS: Intracellular cholesterol-content of cells was modulated and impacts on HEV were monitored using qPCR, Western blot, microscopy, virus-titration and density-gradient centrifugation. Blood-lipids and HEV-RNA were routinely quantified in chronically infected patients during follow-up visits. RESULTS: In HEV-infected cells, decreased levels of cholesterol are found. In patients, HEV infection decreases serum-lipid concentrations. Importantly, statin treatment herein increases viral titers. Similarly, reduction of intracellular cholesterol via simvastatin treatment increases viral release in vitro. On the contrary, elevating intracellular cholesterol via LDL or 25-hydroxycholesterol strongly reduces viral release due to enhanced lysosomal degradation of HEV. Drug-induced elevation of intracellular cholesterol via fenofibrate or PSC833 impairs HEV release via the same mechanism. CONCLUSIONS: This study analyses the crosstalk between HEV and intracellular cholesterol. The results highlight the importance of an intact cholesterol homeostasis for HEV-release and thereby identify a potential target for antiviral strategies. Especially fenofibrate is considered a promising novel antiviral against HEV. Beyond this, the study may help clinicians evaluating co-treatments of HEV-infected patients with statins, as this may be counter indicated

Risk Factors for Hepatitis E Virus Infection and Eating Habits in Kidney Transplant Recipients

There is a significant risk for ongoing and treatment-resistant courses of hepatitis E virus (HEV) infection in patients after solid organ transplantation. The aim of this study was to identify risk factors for the development of hepatitis E, including the dietary habits of patients. We conducted a retrospective single-center study with 59 adult kidney and combined kidney transplant recipients who were diagnosed with HEV infection between 2013 and 2020. The outcomes of HEV infections were analyzed during a median follow-up of 4.3 years. Patients were compared with a control cohort of 251 transplant patients with elevated liver enzymes but without evidence of an HEV infection. Patients’ alimentary exposures during the time before disease onset or diagnosis were assessed. Previous intense immunosuppression, especially treatment with high-dose steroids and rituximab, was a significant risk factor to acquire hepatitis E after solid organ transplantation. Only 11 out of 59 (18.6%) patients reached remission without further ribavirin (RBV) treatment. A total of 48 patients were treated with RBV, of which 19 patients (39.6%) had either viral rebounds after the end of treatment or did not reach viral clearance at all. Higher age (>60 years) and a BMI ≤ 20 kg/m2 were risk factors for RBV treatment failure. Deterioration in kidney function with a drop in eGFR (p = 0.046) and a rise in proteinuria was more common in patients with persistent hepatitis E viremia. HEV infection was associated with the consumption of undercooked pork or pork products prior to infection. Patients also reported processing raw meat with bare hands at home more frequently than the controls. Overall, we showed that the intensity of immunosuppression, higher age, a low BMI and the consumption of undercooked pork meat correlated with the development of hepatitis E