Computergestützte Untersuchung der Struktur, Dynamik und Energetik von Proteininteraktionen

Protein interactions play an essential function in virtually all biological processes. To understand the underlying molecular principles it is important to characterize the structure and dynamics of proteins on an atomic level. In this work, different protein interactions were analyzed with various in silico and in vitro methods. The coordination of activity in biological systems requires the existence of different signal transduction pathways that interact with one another and must be precisely regulated. The Src-family members of tyrosine kinase, which are found at the beginning of a many signal pathways, differ in their physiological function despite their large structural similarity. In previous work, it was shown that the flexibility of the SH3-SH2 domain linker is critical for the relative orientation of the SH3 and SH2 domains. In this thesis, the global effect of the flexibility of the SH3-SH2 linker on the structure and dynamics of Src kinases was analyzed using of molecular dynamics (MD) simulations. For this purpose, the rigid SH3-SH2 linker sequence of inactive Hck kinase structure was replaced with the more flexible linker sequence from Lck. In the MD simulations, this lead to a higher flexibility in comparison to the wild-type Hck, which, in turn, reduced the regulatory domain pair's ability to fixate the kinase domain in the inactive conformation. Furthermore, the exchange of the SH3-SH2 domain linker lead to conformational changes in the activation segment of the kinase domain. The resulting alternative conformation showed similarities to the conformation of the tyrosine kinase Abl when bound to the inhibitor Glivec. Thus, the sequence of the SH3-SH2 linker can not only modulate the kinase activity, but can also influence the ligand binding properties of the active site of Src kinases. This could explain why Lck is, in contrast to other Src kinases, inhibited by Glivec. One possibility to activate the Src kinases is the binding of ligands to the regulatory domains. To characterize the affinity and specificity determinants of the molecular recognition, MD simulations and, subsequently, energetic analyses were performed. The interaction between the Tip protein of Herpesvirus saimiri and the SH3 domain of tyrosine kinase Lyn was chosen as a model system. Tip binds to the SH3 domain by using the sequence motif (175PTPPLPPRPANLG187), which contains a PPII-helix. The role of all non-prolines was analyzed with the help of an in silico alanine scan. The resulting order of importance with respect to the binding, could be experimentally verified using fluorescence spectroscopy. In addition, by taking entropic contributions into account, a modified approach which allows reliable statements about the effect of terminal ligand truncations was developed. By using this strategy, it was shown that the truncation of the Tip peptide by the L186/G187 residues leads to a lower binding af_nity for LynSH3. In contrast, when binding the truncated Tip peptide to homologous SH3 domains, no changes in the binding affinities were detected. It could furthermore be shown that the interaction between L186/G187 in Tip and H41 in LynSH3 significantly increases the binding affinity. The coordinated interplay between kinases and phosphatases represents a fundamental regulatory mechanism in biological systems. Protein phosphatase 1 (PP1), a major serine/threonine phosphatase, regulates its enzymatic activity and substrate specificity by acquisition of different binding partners. These interactions are primarily mediated by a five residue sequence motif, the properties of which are thus far not well characterized. Detailed knowledge of its sequence properties would greatly facilitate the identification of new interaction partners. In this work, the PP1 docking motif was thoroughly characterized with a combination of in silico (molecular modeling, MD simulations) and in vitro (GST pull-down) analysis. It was shown that the amino acid at position +1 represents a key anchor point of the motif, as it forms a network of salt bridges and hydrogen bonds to PP1. In addition, it could be shown that positions +1 and +2 of the docking motif prefer positively charged and/or polar residues. In contrast, the position +3 clearly favors the hydrophobic amino acid valine. The largest sequence variability was observed at position +4, whereas position +5 only tolerates the unpolar and aromatic side chains of phenylalanine and tryptophan. By using a combined approach and performing a systematic analysis of each individual position of the PP11 docking motif, the specific interaction motif [HKR][ACHKMNQRSTV][V][CHKNQRST][FW] could be defined. The usefulness of this motif for genome-wide searches was subsequently tested by identifying and experimentally verifying previously unknown PP1 interactors. The HIV-1 protease, an enzyme of the HI virus, is essential for replication and assembly of the virus. This property makes it an important target for the design of antiviral agents for AIDS treatment. A major problem of the current HIV therapies is the rapid development of resistance to antiretroviral drugs by genetic mutation. It is necessary to understand the resistance mechanism in order to develop more effective and longer lasting treatment regimens. In this thesis, the mutation E35D, which has been described in context with the inhibitor amprenavir, was analyzed. To gain insight into the resistance mechanism, molecular dynamics simulations of the wild-type's as well as the mutant's free, and ligand complexed proteases were performed. In the course of the simulations, an increased flexibility of the mutated HIV protease's flaps, which are parts of the binding pocket, could be observed. This affects the conformational equilibrium between the closed and semiopen conformations of the free protease, whereas in the ligand bound protease some intermolecular interactions are disrupted. Energetic analysis showed that the mutation also causes a significant reduction of the calculated ligand binding energies, thus giving a plausible explanation for its ability to develop resistance. Further analysis showed that the amino acid E35 is also located in an epitope which is important for the recognition of the virus by the immune system. Mutations in the epitope allow the virus to hamper the recognition process, thereby suppressing an immune system response. The results of the investigation suggest that the mutation conveys resistance against protease inhibitors and also allows the virus to escape immune system recognition.Proteininteraktionen spielen praktisch bei allen biologischen Prozessen eine wesentliche Rolle. Um die zugrunde liegenden molekularen Vorgänge zu verstehen, ist es notwendig die Struktur und Dynamik von Proteinen auf atomarer Ebene zu charakterisieren. In der vorliegenden Arbeit wurde eine Reihe von Proteininteraktionen eingehend mit verschiedenen in silico und in vitro Methoden untersucht. Die Koordination von Abläufen in biologischen Systemen erfordert das Vorhandensein verschiedener Signalübertragungswege, die untereinander in Wechselwirkung stehen und exakt reguliert werden müssen. Die Tyrosinkinasen der Src-Familie, die am Anfang vieler Signalwege stehen unterscheiden sich trotz ihrer großen strukturellen Ähnlichkeit in ihren physiologischen Funktionen. In früheren Arbeiten wurde bereits gezeigt, dass die Flexibilität des SH3-SH2 Domänenlinkers kritisch für die relative Orientierung von SH3- und SH2-Domäne ist. Im Rahmen dieser Arbeit wurde mit Hilfe von Moleküldynamik(MD)-Simulationen untersucht, ob die Flexibilität des SH3-SH2 Linkers einen globalen Einfluss auf die Struktur und Dynamik von Src-Kinasen hat. Hierzu wurde die starre SH3-SH2 Linkersequenz aus Hck in der Struktur der inaktiven Kinase durch die flexiblere Linkersequenz aus Lck ersetzt. Dies führte in MD-Simulationen zu einer erhöhten Flexibilität im Vergleich zur Wildtyp-Hck und somit zu einer verringerten Fähigkeit des regulatorischen Domänenpaars die Kinasedomäne in der inaktiven Konformation zu fixieren. Des Weiteren führte der Austausch des SH3-SH2 Domänenlinkers zu konformationellen Änderungen im Aktivierungssegment der Kinasedomäne. Die hierbei eingenommenen alternativen Konformationen zeigten Ähnlichkeiten zur Konformation der Tyrosinkinase Abl, die nach Bindung des Inhibitors Glivec eingenommen wird. Die Sequenz des SH3-SH2 Linkers könnte demzufolge nicht nur die Modulation der Kinaseaktivität, sondern auch die Ligandenbindungseigenschaften im aktiven Zentrum von Src-Kinasen beeinflussen. Dies könnte erklären, warum Lck im Gegensatz zu den anderen Src-Kinasen durch Glivec gehemmt werden kann. Die Aktivierung der Src-Kinasen kann unter anderem durch die Bindung von Liganden an die regulatorischen Domänen vermittelt werden. Zur Charakterisierung der Determinanten der Affinität und Spezifität der molekularen Erkennung wurden MD-Simulationen und darauf aufbauende energetische Analysen durchgeführt. Als Modellsystem wurde hier die Wechselwirkung zwischen dem Tip-Protein aus Herpesvirus saimiri und der SH3-Domäne der Tyrosinkinase Lyn gewählt. Tip bindet mit dem Sequenzmotiv (175PTPPLPPRPANLG187), welches eine PPII-Helix beinhaltet, an die SH3-Domäne. Mit Hilfe eines in silico Alanin-Scans wurde die Rolle aller Nicht-Proline bei der LynSH3-Bindung analysiert. Die dabei erhaltene Reihenfolge der Wichtigkeit für die Bindung konnte anschließend experimentell mittels Fluoreszenzspektroskopischer Messungen bestätigt werden. Außerdem konnte ein modifizierter Ansatz entwickelt werden, der durch die Berücksichtigung entropischer Beiträge auch für die Deletion von terminalen Aminosäuren zuverlässige Aussagen über Bindungsaffinitäten liefert. Mit dieser Strategie konnte gezeigt werden, dass die Verkürzung des Tip-Peptids um die Reste L186/G187 bei der LynSH3-Bindung zu einer schlechteren Bindung führt, wohingegen bei homologen SH3-Domänen kein Effekt beobachtet wurde. Es konnte weiter gezeigt werden, dass die Wechselwirkungen zwischen L186/G187 in Tip und H41 in LynSH3 maßgeblich zu einer Steigerung der Bindungsaffinität beitragen. Das koordinierte Zusammenspiel zwischen Kinasen und Phosphatasen stellt einen wichtigen regulatorischen Mechanismus in biologischen Systemen dar. Die Proteinphosphatase 1 (PP1), eine der bedeutendsten Serin-/Threonin-Phosphatasen, erreicht ihre enzymatische Aktivität und Substratspezifität durch die Bindung verschiedener Interaktionspartner. Diese Interaktion wird primär über ein fünf Aminosäuren langes Sequenzmotiv vermittelt, dessen Eigenschaften bislang nur unzureichend charakterisiert waren, was die Identifizierung neuer Wechselwirkungspartner erschwerte. In der vorliegenden Arbeit wurde das PP1-Bindemotiv mit Hilfe von in silico (Modelling und MD) und in vitro (GST-Pulldown) Analysen eingehend charakterisiert. Es konnte gezeigt werden, dass die Aminosäure an Position +1 einen Ankerpunkt repräsentiert, da sie ein Netzwerk von Salz- und Wasserstoffbrücken zur PP11 bildet. Des Weiteren konnte gezeigt werden, dass die Positionen +1 und +2 im Bindemotiv positiv geladene bzw. polare Aminosäuren tolerieren. Im Gegensatz dazu ließ die Position +3 eine deutliche Präferenz für Valin erkennen. Die größte Variabilität innerhalb des Bindemotivs konnte an der Position +4 beobachtet werden, wohingegen an Position +5 nur die Seitenketten der unpolaren und aromatischen Aminosäuren Phenylalanin und Tryptophan toleriert wurden. Durch den kombinierten Ansatz und die systematische Analyse jeder einzelnen Position des PP1-Bindemotivs war es somit möglich ein spezifisches Interaktionsmotiv [HKR][ACHKMNQRSTV][V][CHKNQRST][FW] aufzustellen. Die Brauchbarkeit dieses Motivs für genomweite Suchen konnte anschließend durch die Identifizierung und experimentelle Verifizierung neuer PP1-Interaktionspartner bestätigt werden. Die HIV-1 Protease, ein Enzym des HI-Virus, ist essentiell für die Bildung reifer Viruspartikel. Somit stellt die Protease ein äußerst wichtiges Zielmolekül für die Entwicklung von Wirkstoffen gegen die Immunschwächekrankheit AIDS dar. Eines der Hauptprobleme der antiretroviralen Therapie ist die schnelle Entwicklung von Resistenzen gegen die eingesetzten Wirkstoffe, welche häufig auf Mutationen der HIV-1 Protease zurückzuführen sind. Zur Entwicklung effektiver und lang anhaltender Wirkstoffe ist es wichtig die Resistenzmechanismen zu verstehen. In dieser Arbeit wurde die Mutation E35D, welche unter anderem im Zusammenhang mit dem Inhibitor Amprenavir beschrieben wird, untersucht. Um einen Einblick in den Resistenzmechanismus zu erhalten, wurden sowohl für den Wildtyp als auch für die Mutante MD-Simulationen der freien und ligandengebundenen Protease durchgeführt. Im Verlauf der Simulationen wurde für die mutierte HIV-1 Protease eine höhere Flexibilität von Schleifenbereichen sichtbar, die Teil der Ligandenbindungstasche sind. Dies wirkte sich bei der freien Protease auf das Gleichgewicht zwischen geschlossener und halboffener Konformation aus, wohingegen bei der ligandengebundenen Protease intermolekulare Wechselwirkungen gestört wurden. Energetische Analysen zeigten, dass die Mutation zu einer signifikanten Erniedrigung der Bindungsenergien für Liganden führt und somit die beobachtete Resistenzentstehung erklären kann. Weiterführende Analysen zeigten, dass die Aminosäure E35 auch in einem Epitop liegt, das für die Erkennung des Virus durch das Immunsystem wichtig ist. Das Virus kann durch Mutationen im Epitop den Erkennungsprozess erschweren und so zu einer Unterdrückung der Immunantwort führen. Die Ergebnisse der Untersuchungen deuten darauf hin, dass die Mutation sowohl Resistenz gegen Proteaseinhibitoren vermittelt als auch eine Flucht vor der Erkennung durch das Immunsystem ermöglicht

Disturbed neuronal ER-Golgi sorting of unassembled glycine receptors suggests altered subcellular processing is a cause of human hyperekplexia.

Recent studies on the pathogenic mechanisms of recessive hyperekplexia indicate disturbances in glycine receptor (GlyR) α1 biogenesis. Here, we examine the properties of a range of novel glycine receptor mutants identified in human hyperekplexia patients using expression in transfected cell lines and primary neurons. All of the novel mutants localized in the large extracellular domain of the GlyR α1 have reduced cell surface expression with a high proportion of receptors being retained in the ER, although there is forward trafficking of glycosylated subpopulations into the ER-Golgi intermediate compartment and cis-Golgi compartment. CD spectroscopy revealed that the mutant receptors have proportions of secondary structural elements similar to wild-type receptors. Two mutants in loop B (G160R, T162M) were functional, but none of those in loop D/β2-3 were. One nonfunctional truncated mutant (R316X) could be rescued by coexpression with the lacking C-terminal domain. We conclude that a proportion of GlyR α1 mutants can be transported to the plasma membrane but do not necessarily form functional ion channels. We suggest that loop D/β2-3 is an important determinant for GlyR trafficking and functionality, whereas alterations to loop B alter agonist potencies, indicating that residues here are critical elements in ligand binding.This work was supported by the Deutsche Forschungsgemeinschaft (Grant DFG VI586 to C.V.) and the European Union (FP7 project Neurocypres to C.J.K., K.L.P., and S.C.R.L.). N. Schaefer and G.L. are supported by the GSLS Wuerzburg. S.C.R.L. is a Wellcome Trust Senior Research Fellow in Basic Biomedical Research.This is the author accepted manuscript. The final version is available from the Society of Neuroscience via http://dx.doi.org/10.1523/JNEUROSCI.1509-14.201

ELM—the database of eukaryotic linear motifs

Linear motifs are short, evolutionarily plastic components of regulatory proteins and provide low-affinity interaction interfaces. These compact modules play central roles in mediating every aspect of the regulatory functionality of the cell. They are particularly prominent in mediating cell signaling, controlling protein turnover and directing protein localization. Given their importance, our understanding of motifs is surprisingly limited, largely as a result of the difficulty of discovery, both experimentally and computationally. The Eukaryotic Linear Motif (ELM) resource at http://elm.eu.org provides the biological community with a comprehensive database of known experimentally validated motifs, and an exploratory tool to discover putative linear motifs in user-submitted protein sequences. The current update of the ELM database comprises 1800 annotated motif instances representing 170 distinct functional classes, including approximately 500 novel instances and 24 novel classes. Several older motif class entries have been also revisited, improving annotation and adding novel instances. Furthermore, addition of full-text search capabilities, an enhanced interface and simplified batch download has improved the overall accessibility of the ELM data. The motif discovery portion of the ELM resource has added conservation, and structural attributes have been incorporated to aid users to discriminate biologically relevant motifs from stochastically occurring non-functional instance

Development and Validation of a Prediction Model for Future Estimated Glomerular Filtration Rate in People With Type 2 Diabetes and Chronic Kidney Disease

Importance: Type 2 diabetes increases the risk of progressive diabetic kidney disease, but reliable prediction tools that can be used in clinical practice and aid in patients' understanding of disease progression are currently lacking. Objective: To develop and externally validate a model to predict future trajectories in estimated glomerular filtration rate (eGFR) in adults with type 2 diabetes and chronic kidney disease using data from 3 European multinational cohorts. Design, Setting, and Participants: This prognostic study used baseline and follow-up information collected between February 2010 and December 2019 from 3 prospective multinational cohort studies: PROVALID (Prospective Cohort Study in Patients with Type 2 Diabetes Mellitus for Validation of Biomarkers), GCKD (German Chronic Kidney Disease), and DIACORE (Diabetes Cohorte). A total of 4637 adult participants (aged 18-75 years) with type 2 diabetes and mildly to moderately impaired kidney function (baseline eGFR of ≥30 mL/min/1.73 m2) were included. Data were analyzed between June 30, 2021, and January 31, 2023. Main Outcomes and Measures: Thirteen variables readily available from routine clinical care visits (age, sex, body mass index; smoking status; hemoglobin A1c[mmol/mol and percentage]; hemoglobin, and serum cholesterol levels; mean arterial pressure, urinary albumin-creatinine ratio, and intake of glucose-lowering, blood-pressure lowering, or lipid-lowering medication) were selected as predictors. Repeated eGFR measurements at baseline and follow-up visits were used as the outcome. A linear mixed-effects model for repeated eGFR measurements at study entry up to the last recorded follow-up visit (up to 5 years after baseline) was fit and externally validated. Results: Among 4637 adults with type 2 diabetes and chronic kidney disease (mean [SD] age at baseline, 63.5 [9.1] years; 2680 men [57.8%]; all of White race), 3323 participants from the PROVALID and GCKD studies (mean [SD] age at baseline, 63.2 [9.3] years; 1864 men [56.1%]) were included in the model development cohort, and 1314 participants from the DIACORE study (mean [SD] age at baseline, 64.5 [8.3] years; 816 men [62.1%]) were included in the external validation cohort, with a mean (SD) follow-up of 5.0 (0.6) years. Updating the random coefficient estimates with baseline eGFR values yielded improved predictive performance, which was particularly evident in the visual inspection of the calibration curve (calibration slope at 5 years: 1.09; 95% CI, 1.04-1.15). The prediction model had good discrimination in the validation cohort, with the lowest C statistic at 5 years after baseline (0.79; 95% CI, 0.77-0.80). The model also had predictive accuracy, with an R2ranging from 0.70 (95% CI, 0.63-0.76) at year 1 to 0.58 (95% CI, 0.53-0.63) at year 5. Conclusions and Relevance: In this prognostic study, a reliable prediction model was developed and externally validated; the robust model was well calibrated and capable of predicting kidney function decline up to 5 years after baseline. The results and prediction model are publicly available in an accompanying web-based application, which may open the way for improved prediction of individual eGFR trajectories and disease progression.</p

A prediction model for the decline in renal function in people with type 2 diabetes mellitus: study protocol

Background Chronic kidney disease (CKD) is a well-established complication in people with diabetes mellitus. Roughly one quarter of prevalent patients with diabetes exhibit a CKD stage of 3 or higher and the individual course of progression is highly variable. Therefore, there is a clear need to identify patients at high risk for fast progression and the implementation of preventative strategies. Existing prediction models of renal function decline, however, aim to assess the risk by artificially grouped patients prior to model building into risk strata defined by the categorization of the least-squares slope through the longitudinally fluctuating eGFR values, resulting in a loss of predictive precision and accuracy. Methods This study protocol describes the development and validation of a prediction model for the longitudinal progression of renal function decline in Caucasian patients with type 2 diabetes mellitus (DM2). For development and internal-external validation, two prospective multicenter observational studies will be used (PROVALID and GCKD). The estimated glomerular filtration rate (eGFR) obtained at baseline and at all planned follow-up visits will be the longitudinal outcome. Demographics, clinical information and laboratory measurements available at a baseline visit will be used as predictors in addition to random country-specific intercepts to account for the clustered data. A multivariable mixed-effects model including the main effects of the clinical variables and their interactions with time will be fitted. In application, this model can be used to obtain personalized predictions of an eGFR trajectory conditional on baseline eGFR values. The final model will then undergo external validation using a third prospective cohort (DIACORE). The final prediction model will be made publicly available through the implementation of an R shiny web application. Discussion Our proposed state-of-the-art methodology will be developed using multiple multicentre study cohorts of people with DM2 in various CKD stages at baseline, who have received modern therapeutic treatment strategies of diabetic kidney disease in contrast to previous models. Hence, we anticipate that the multivariable prediction model will aid as an additional informative tool to determine the patient-specific progression of renal function and provide a useful guide to early on identify individuals with DM2 at high risk for rapid progression

Expectation of clinical decision support systems: a survey study among nephrologist end-users

Background Chronic kidney disease (CKD), a major public health problem with differing disease etiologies, leads to complications, comorbidities, polypharmacy, and mortality. Monitoring disease progression and personalized treatment efforts are crucial for long-term patient outcomes. Physicians need to integrate different data levels, e.g., clinical parameters, biomarkers, and drug information, with medical knowledge. Clinical decision support systems (CDSS) can tackle these issues and improve patient management. Knowledge about the awareness and implementation of CDSS in Germany within the field of nephrology is scarce. Purpose Nephrologists’ attitude towards any CDSS and potential CDSS features of interest, like adverse event prediction algorithms, is important for a successful implementation. This survey investigates nephrologists’ experiences with and expectations towards a useful CDSS for daily medical routine in the outpatient setting. Methods The 38-item questionnaire survey was conducted either by telephone or as a do-it-yourself online interview amongst nephrologists across all of Germany. Answers were collected and analysed using the Electronic Data Capture System REDCap, as well as Stata SE 15.1, and Excel. The survey consisted of four modules: experiences with CDSS (M1), expectations towards a helpful CDSS (M2), evaluation of adverse event prediction algorithms (M3), and ethical aspects of CDSS (M4). Descriptive statistical analyses of all questions were conducted. Results The study population comprised 54 physicians, with a response rate of about 80–100% per question. Most participants were aged between 51–60 years (45.1%), 64% were male, and most participants had been working in nephrology out-patient clinics for a median of 10.5 years. Overall, CDSS use was poor (81.2%), often due to lack of knowledge about existing CDSS. Most participants (79%) believed CDSS to be helpful in the management of CKD patients with a high willingness to try out a CDSS. Of all adverse event prediction algorithms, prediction of CKD progression (97.8%) and in-silico simulations of disease progression when changing, e. g., lifestyle or medication (97.7%) were rated most important. The spectrum of answers on ethical aspects of CDSS was diverse. Conclusion This survey provides insights into experience with and expectations of out-patient nephrologists on CDSS. Despite the current lack of knowledge on CDSS, the willingness to integrate CDSS into daily patient care, and the need for adverse event prediction algorithms was high.Open Access funding enabled and organized by Projekt DEAL.Universitätsklinikum Freiburg (8975

Prevalence of hereditary tubulointerstitial kidney diseases in the German Chronic Kidney Disease study

Hereditary chronic kidney disease (CKD) appears to be more frequent than the clinical perception. Exome sequencing (ES) studies in CKD cohorts could identify pathogenic variants in ~10% of individuals. Tubulointerstitial kidney diseases, showing no typical clinical/histologic finding but tubulointerstitial fibrosis, are particularly difficult to diagnose. We used a targeted panel (29 genes) and MUC1 -SNaPshot to sequence 271 DNAs, selected in defined disease entities and age cutoffs from 5217 individuals in the German Chronic Kidney Disease cohort. We identified 33 pathogenic variants. Of these 27 (81.8%) were in COL4A3/4/5 , the largest group being 15 COL4A5 variants with nine unrelated individuals carrying c.1871G>A, p.(Gly624Asp). We found three cysteine variants in UMOD , a novel missense and a novel splice variant in HNF1B and the homoplastic MTTF variant m.616T>C. Copy-number analysis identified a heterozygous COL4A5 deletion, and a HNF1B duplication/deletion, respectively. Overall, pathogenic variants were present in 12.5% (34/271) and variants of unknown significance in 9.6% (26/271) of selected individuals. Bioinformatic predictions paired with gold standard diagnostics for MUC1 (SNaPshot) could not identify the typical cytosine duplication (“c.428dupC”) in any individual, implying that ADTKD- MUC1 is rare. Our study shows that >10% of selected individuals carry disease-causing variants in genes partly associated with tubulointerstitial kidney diseases. COL4A3/4/5 genes constitute the largest fraction, implying they are regularly overlooked using clinical Alport syndrome criteria and displaying the existence of phenocopies. We identified variants easily missed by some ES pipelines. The clinical filtering criteria applied enriched for an underlying genetic disorder.Deutsche Forschungsgemeinschaft (German Research Foundation) https://doi.org/10.13039/50110000165

Apolipoprotein A-IV concentrations and cancer in a large cohort of chronic kidney disease patients: results from the GCKD study

Background Chronic kidney disease (CKD) is highly connected to inflammation and oxidative stress. Both favour the development of cancer in CKD patients. Serum apolipoprotein A-IV (apoA-IV) concentrations are influenced by kidney function and are an early marker of kidney impairment. Besides others, it has antioxidant and anti-inflammatory properties. Proteomic studies and small case–control studies identified low apoA-IV as a biomarker for various forms of cancer; however, prospective studies are lacking. We therefore investigated whether serum apoA-IV is associated with cancer in the German Chronic Kidney Disease (GCKD) study. Methods These analyses include 5039 Caucasian patients from the prospective GCKD cohort study followed for 6.5 years. Main inclusion criteria were an eGFR of 30–60 mL/min/1.73m2 or an eGFR > 60 mL/min/1.73m2 in the presence of overt proteinuria. Results Mean apoA-IV concentrations of the entire cohort were 28.9 ± 9.8 mg/dL (median 27.6 mg/dL). 615 patients had a history of cancer before the enrolment into the study. ApoA-IV concentrations above the median were associated with a lower odds for a history of cancer (OR = 0.79, p = 0.02 when adjusted age, sex, smoking, diabetes, BMI, albuminuria, statin intake, and eGFRcreatinine). During follow-up 368 patients developed an incident cancer event and those with apoA-IV above the median had a lower risk (HR = 0.72, 95%CI 0.57–0.90, P = 0.004). Finally, 62 patients died from such an incident cancer event and each 10 mg/dL higher apoA-IV concentrations were associated with a lower risk for fatal cancer (HR = 0.62, 95%CI 0.44–0.88, P = 0.007). Conclusions Our data indicate an association of high apoA-IV concentrations with reduced frequencies of a history of cancer as well as incident fatal and non-fatal cancer events in a large cohort of patients with CKD

Association of mineral and bone biomarkers with adverse cardiovascular outcomes and mortality in the German Chronic Kidney Disease (GCKD) cohort

Mineral and bone disorder (MBD) in chronic kidney disease (CKD) is tightly linked to cardiovascular disease (CVD). In this study, we aimed to compare the prognostic value of nine MBD biomarkers to determine those associated best with adverse cardiovascular (CV) outcomes and mortality. In 5 217 participants of the German CKD (GCKD) study enrolled with an estimated glomerular filtration rate (eGFR) between 30–60 mL·min −1 per 1.73 m 2 or overt proteinuria, serum osteoprotegerin (OPG), C-terminal fibroblast growth factor-23 (FGF23), intact parathyroid hormone (iPTH), bone alkaline phosphatase (BAP), cross-linked C-telopeptide of type 1 collagen (CTX1), procollagen 1 intact N-terminal propeptide (P1NP), phosphate, calcium, and 25-OH vitamin D were measured at baseline. Participants with missing values among these parameters ( n  = 971) were excluded, leaving a total of 4 246 participants for analysis. During a median follow-up of 6.5 years, 387 non-CV deaths, 173 CV deaths, 645 nonfatal major adverse CV events (MACEs) and 368 hospitalizations for congestive heart failure (CHF) were observed. OPG and FGF23 were associated with all outcomes, with the highest hazard ratios (HRs) for OPG. In the final Cox regression model, adjusted for CV risk factors, including kidney function and all other investigated biomarkers, each standard deviation increase in OPG was associated with non-CV death (HR 1.76, 95% CI: 1.35–2.30), CV death (HR 2.18, 95% CI: 1.50–3.16), MACE (HR 1.38, 95% CI: 1.12–1.71) and hospitalization for CHF (HR 2.05, 95% CI: 1.56–2.69). Out of the nine biomarkers examined, stratification based on serum OPG best identified the CKD patients who were at the highest risk for any adverse CV outcome and mortality.Else Kröner-Fresenius-Stiftung (Else Kroner-Fresenius Foundation) https://doi.org/10.13039/501100003042German Society of Internal Medicine 361357Bundesministerium für Bildung und Forschung (Federal Ministry of Education and Research) https://doi.org/10.13039/501100002347KfH (Kuratorium für Heimdialyse und Nierentransplantation e.V.—Stiftung Präventivmedizin- no grant numberDeutsche Forschungsgemeinschaft (German Research Foundation) https://doi.org/10.13039/50110000165

Association of mineral and bone biomarkers with adverse cardiovascular outcomes and mortality in the German Chronic Kidney Disease (GCKD) cohort

Mineral and bone disorder (MBD) in chronic kidney disease (CKD) is tightly linked to cardiovascular disease (CVD). In this study, we aimed to compare the prognostic value of nine MBD biomarkers to determine those associated best with adverse cardiovascular (CV) outcomes and mortality. In 5 217 participants of the German CKD (GCKD) study enrolled with an estimated glomerular filtration rate (eGFR) between 30-60 mL·min per 1.73 m or overt proteinuria, serum osteoprotegerin (OPG), C-terminal fibroblast growth factor-23 (FGF23), intact parathyroid hormone (iPTH), bone alkaline phosphatase (BAP), cross-linked C-telopeptide of type 1 collagen (CTX1), procollagen 1 intact N-terminal propeptide (P1NP), phosphate, calcium, and 25-OH vitamin D were measured at baseline. Participants with missing values among these parameters (n = 971) were excluded, leaving a total of 4 246 participants for analysis. During a median follow-up of 6.5 years, 387 non-CV deaths, 173 CV deaths, 645 nonfatal major adverse CV events (MACEs) and 368 hospitalizations for congestive heart failure (CHF) were observed. OPG and FGF23 were associated with all outcomes, with the highest hazard ratios (HRs) for OPG. In the final Cox regression model, adjusted for CV risk factors, including kidney function and all other investigated biomarkers, each standard deviation increase in OPG was associated with non-CV death (HR 1.76, 95% CI: 1.35-2.30), CV death (HR 2.18, 95% CI: 1.50-3.16), MACE (HR 1.38, 95% CI: 1.12-1.71) and hospitalization for CHF (HR 2.05, 95% CI: 1.56-2.69). Out of the nine biomarkers examined, stratification based on serum OPG best identified the CKD patients who were at the highest risk for any adverse CV outcome and mortality