Einfluss genetischer Varianten im Aquaporin 5-Promotor-Gen auf Schlüsselmechanismen der Sepsis

Die Sepsis stellt eine der häufigsten Komplikationen auf Intensivstationen dar und die Letalität ist mit 30 bis 50 % noch immer sehr hoch. Intraindividuelle Unterschiede in Krankheitsverlauf und Letalität können durch klassische Risikofaktoren nicht allein erklärt werden, vielmehr scheinen auch genetische Faktoren eine Rolle zu spielen. Da das C-Allel des funktionellen Aquaporin 5 (AQP5) A(-1364)C –Promotorpolymorphismus sowohl mit einer verringerten AQP5-Expression als auch mit einem verbesserten Überleben bei schwerer Sepsis einhergeht, scheint AQP5 ein Schlüsselprotein in der Sepsis darzustellen. Unklar war allerdings über welchen Mechanismus Genotyp, Aquaporinexpression und Letalität der schweren Sepsis miteinander verknüpft sind. Ziel dieser Arbeit war es daher aufzuklären, 1) über welchen Mechanismus der Polymorphismus die AQP5-Expression verändert und 2) wie die AQP5-Expression Mechanismen der Sepsis beeinflusst. Zur Beantwortung der ersten Frage wurde zum einen untersucht, ob der Polymorphismus zu einer unterschiedlichen Transkriptionsfaktorenbindung führt und ob der Polymorphismus das Methylierungsmuster der DNA im Vollblut septischer Patienten beeinflusst. Eine in silico- Analyse ergab, dass die Tanskriptionsfaktoren NMP4, c-Myb und NFAT putative Bindungsstellen um Position -1364 tragen. Mittels Electrophoretic mobility Shift Assays (EMSA) konnte eine spezifische Bindung von NMP4 verifiziert werden. Diese scheint beim C-Allel stärker ausgeprägt zu sein. Reporterassays zeigten jedoch, dass NMP4 die Promotoraktivität des AQP5-Promotors unabhängig vom Genotyp erhöht. Auch die AQP5 mRNA-Expression wurde in HEK293 Zellen signifikant durch NMP4 erhöht, wie Real-Time-PCR-Experimente zeigten. Außerdem konnte eine neue Bindungsstelle für NMP4 in der AQP5-Promotoregion von –nt592/-602 identifiziert werden, die für die starke Erhöhung der AQP5-Promotoraktivität durch NMP4 mitverantwortlich zu sein scheint. Da NMP4 also zwar einen transkriptionellen Regulator der AQP5-Expression darstellt, nicht aber die unterschiedliche AQP5-Expression bei A- und C-Allelträgern bedingt, wurde weiter untersucht, ob der Polymorphismus Einfluss auf das Methylierungsmuster hat. Hierbei konnte im Vollblut von septischen Patienten 4 Positionen (nt-1036, nt-1014, nt-856 und nt-839) detektiert werden, in denen C-Allelträger eine signifikant erhöhte Methylierung aufwiesen. Ob dieser Unterschied tatsächlich für die unterschiedliche AQP5-Expression verantwortlich ist, konnte abschließend nicht geklärt werden. Dafür spricht aber, dass eine verringerte AQP5-Promotormethylierung mit einer erhöhten AQP5-Expression in Immunzellen einhergeht. Ferner wird die Methylierung von Immunzellen durch LPS vermindert. Dieses führte zu einer verstärkten AQP5-Expression in THP-1 Zellen. Dieses untermauert die Stimulation der Zellen mit LPS, welche demonstrierte, dass die AQP5-Expression in THP-1 Zellen durch LPS verändert wird. Ein weiterer wichtiger Punkt dieser Arbeit war die Frage, ob AQP5 ein Schlüsselprotein in der LPS induzierten Inflammation darstellt. Hierzu wurden AQP5-KO Mäusen sowie deren WT Wurfgeschwistern LPS injiziert und deren Überlebenswahrscheinlichkeit untersucht. Es konnte beobachtet werden, dass die KO-Mäuse im Vergleich zu den WT-Mäusen eine bessere Überlebensrate aufwiesen. Diese in vivo Daten wurden auch in vitro verifiziert. Ziel war es aufzuklären, wie AQP5 Mechanismen der Sepsis beeinflusst. Zunächst konnte mittels immunologischer Färbung nachgewiesen werden, dass C-Allelträger eine verringerte AQP5-Expression auf neutrophilen Granulozyten aufweisen. Dieses ging mit einer verminderten Migration dieser Zellen einher. In Übereinstimmung damit zeigen AQP5-überexprimierte Jurkat Zellen eine verstärkte Migration im Vergleich zu nativen Zellen. Demgegenüber unterschied sich die Proliferation monozytärer Zellen nicht zwischen den Genotypen. Nachdem wir zeigen konnten, dass die AQP5-Expression mit der Sepsisletalität assoziiert ist, wurde untersucht, ob die LPS induzierte Änderung der AQP5-Expression durch Therapeutika beeinflussbar ist. Hierfür wurden THP-1 Zellen mit LPS und cAMP stimuliert. Die Daten zeigten jedoch, dass cAMP LPS induzierte Effekte nicht aufheben kann. In folgenden Studien bleibt nun zu klären, wie LPS die Signalkaskade der AQP5-Expression und Translokation beeinflusst. Zusammenfassend scheint AQP5 ein Schlüsselprotein in der Sepsis zu sein, indem es die Migration von neutrophilen Granulozyten beeinflusst. Auch die Methylierung am AQP5-Promotor spielt in der Sepsis und möglicherweise auch bei der unterschiedlichen Expression von A- und C-Allelträgern eine Rolle.Sepsis is one of the most common complications at intensive care units in Germany and lethality remains with 30 – 50 % unbounded high. Wide variability exists regarding the outcome in severe sepsis, which in part may be caused by genetic variations. A potential candidate for such variations is the gene-encoding aquaporin (AQP) 5, which mediates key mechanisms of inflammation that prevail in sepsis, including cell migration and proliferation. In addition the C allele of the functional AQP5 A(-1364)C promotor polymorphism is associated with decreased AQP5 expression but better outcome in severe sepsis. So far the underlying molecular and physiologic alterations linking decreased AQP5 expression to increased 30-day survival have not proven. Therefore the objective of the present thesis is to elucidate 1) the molecular mechanisms which facilitate the different AQP5 expression in A- und C genotypes and 2) the underlying mechanism of how AQP5 expression affects sepsis survival. Binding of transcription factors to the direct promoter region of the polymorphism and AQP5 promoter methylation of septic patients are analyzed in order to answer the first question. In silico analysis reveals NMP4, c-Myb and NFAT as putative transcription factor candidates. Electrophoretic mobility shift assays (EMSAs) confirm specific binding of NMP4 to the promotor region of nt-1364, thereby binding to the C- allele appears to be stronger. In contrast to this, reporter assays show increased promotor activity of all analyzed promoter constructs irrespective of genotype. In addition real-time-PCR shows increased AQP5 expression after NMP4 overexpression in HEK293 cells. Furthermore an additional NMP4 binding site can be identified in the AQP5 promoter at position –nt592/-602. Epigenetic regulation by DNA methylation is considered as a second possible mechanism. This is investigated in whole blood samples of septic patients. Four positions with increased methylation in C-allele patients can be detected (nt-1036, nt-1014, nt-856 and nt-839). The influence of decreased methylation in these specific positons on AQP5 expression cannot be elucidated. But it can be shown that decreased AQP5 promoter methylation is associated with increased AQP5 expression in immune cells. Therefore the decreased AQP5 expression of C-allele genotypes could be caused by increased methylation of these positions. Furthermore Lipopolysaccharide (LPS) alters AQP5 promoter methylation. This can be demonstrated in a monocytic cell line, which was stimulated with LPS. The second part of this thesis is to investigate the mechanisms of AQP5 protein expression on key mechanisms of sepsis. First of all we investigate the survival of AQP5-Knockout (KO) mice and wildtyp mice after LPS induced inflammation. AQP5-KO mice show significantly increased survival compared to wildtyp mice. These data are verified in vitro. It is investigated if AQP5 expression influences immune cell migration or proliferation. It can be demonstrated that neutrophil granulocytes from AA-genotypes have increased AQP5 expression and show increased neutrophil migration compared to AC/CC-genotypes. In addition AQP5 overexpression increases migration in the lymphocytic cell line Jurkat. In contrast proliferation of monocytic cells does not differ between genotypes. Finally, we investigate if the downstream effector of the β2 adrenergic signaling pathway cAMP is a therapeutic option in sepsis treatment. cAMP does not abolish LPS induced effects on AQP5 expression or morphology of THP-1 cells. Therefore β2 adrenergic stimulation is not a therapeutic option in sepsis treatment. Summarizing AQP5 seems to be a key mediator in sepsis as AQP5 expression modulates sepsis survival and migration of neutrophil granulocytes. In addition, a novel transcriptional regulator of AQP5 expression can be identified as NMP4. Its role in sepsis has to be analyzed by prospective studies. One important regulatory mechanism of AQP5 expression in sepsis might be AQP5 promoter methylation

Etiological diagnosis in limb reduction defects and the number of affected limbs:A population-based study in the Northern Netherlands

Limb reduction defects (LRDs) that affect multiple limbs are considered to be more often heritable, but only few studies have substantiated this. We aimed to investigate if an etiological diagnosis (genetic disorder or clinically recognizable disorder) is more likely to be made when multiple limbs are affected compared to when only one limb is affected. We used data from EUROCAT Northern Netherlands and included 391 fetuses and children with LRDs born in 1981-2017. Cases were classified as having a transverse, longitudinal (preaxial/postaxial/central/mixed), intercalary, or complex LRD of one or more limbs and as having an isolated LRD or multiple congenital anomalies (MCA). We calculated the probability of obtaining an etiological diagnosis in cases with multiple affected limbs versus one affected limb using relative risk (RR) scores and Fisher's exact test. We showed that an etiological diagnosis was made three times more often when an LRD occurred in multiple limbs compared to when it occurred in one limb (RR 2.9, 95% CI 2.2-3.8, p <0.001). No genetic disorders were found in isolated cases with only one affected limb, whereas a genetic disorder was identified in 16% of MCA cases with one affected limb. A clinically recognizable disorder was found in 47% of MCA cases with one affected limb. Genetic counseling rates were similar. We conclude that reduction defects of multiple limbs are indeed more often heritable. Genetic testing seems less useful in isolated cases with one affected limb, but is warranted in MCA cases with one affected limb

Hypoxia Inducible Factor-2Alpha and Prolinhydroxylase 2 Polymorphisms in Patients with Acute Respiratory Distress Syndrome (ARDS)

Hypoxia-inducible-factor-2 alpha (HIF-2 alpha) and HIF-2 degrading prolyl-hydroxylases (PHD) are key regulators of adaptive hypoxic responses i.e., in acute respiratory distress syndrome (ARDS). Specifically, functionally active genetic variants of HIF-2 alpha (single nucleotide polymorphism (SNP) [ch2:46441523(hg18)]) and PHD2 (C/T;SNP rs516651 and T/C;SNP rs480902) are associated with improved adaptation to hypoxia i.e., in high-altitude residents. However, little is known about these SNPs' prevalence in Caucasians and impact on ARDS-outcome. Thus, we tested the hypotheses that in Caucasian ARDS patients SNPs in HIF-2 alpha or PHD2 genes are (1) common, and (2) independent risk factors for 30-day mortality. After ethics-committee approval, 272 ARDS patients were prospectively included, genotyped for PHD2 (Taqman SNP Genotyping Assay) and HIF-2 alpha-polymorphism (restriction digest + agarose-gel visualization), and genotype dependent 30-day mortality was analyzed using Kaplan-Meier-plots and multivariate Cox-regression analyses. Frequencies were 99.62% for homozygous HIF-2 alpha CC-carriers (CG: 0.38%;GG: 0%), 2.3% for homozygous PHD2 SNP rs516651 TT-carriers (CT: 18.9%;CC: 78.8%), and 3.7% for homozygous PHD2 SNP rs480902 TT-carriers (CT: 43.9%;CC: 52.4%). PHD2 rs516651 TT-genotype in ARDS was independently associated with a 3.34 times greater mortality risk (OR 3.34, CI 1.09-10.22;p = 0.034) within 30-days, whereas the other SNPs had no significant impact (p = ns). The homozygous HIF-2 alpha GG-genotype was not present in our Caucasian ARDS cohort;however PHD2 SNPs exist in Caucasians, and PHD2 rs516651 TT-genotype was associated with an increased 30-day mortality suggesting a relevance for adaptive responses in ARDS

Large scale multifactorial likelihood quantitative analysis of BRCA1 and BRCA2 variants: An ENIGMA resource to support clinical variant classification

The multifactorial likelihood analysis method has demonstrated utility for quantitative assessment of variant pathogenicity for multiple cancer syndrome genes. Independent data types currently incorporated in the model for assessing BRCA1 and BRCA2 variants include clinically calibrated prior probability of pathogenicity based on variant location and bioinformatic prediction of variant effect, co-segregation, family cancer history profile, co-occurrence with a pathogenic variant in the same gene, breast tumor pathology, and case-control information. Research and clinical data for multifactorial likelihood analysis were collated for 1,395 BRCA1/2 predominantly intronic and missense variants, enabling classification based on posterior probability of pathogenicity for 734 variants: 447 variants were classified as (likely) benign, and 94 as (likely) pathogenic; and 248 classifications were new or considerably altered relative to ClinVar submissions. Classifications were compared with information not yet included in the likelihood model, and evidence strengths aligned to those recommended for ACMG/AMP classification codes. Altered mRNA splicing or function relative to known nonpathogenic variant controls were moderately to strongly predictive of variant pathogenicity. Variant absence in population datasets provided supporting evidence for variant pathogenicity. These findings have direct relevance for BRCA1 and BRCA2 variant evaluation, and justify the need for gene-specific calibration of evidence types used for variant classification

Large scale multifactorial likelihood quantitative analysis of BRCA1 and BRCA2 variants: An ENIGMA resource to support clinical variant classification

Abstract The multifactorial likelihood analysis method has demonstrated utility for quantitative assessment of variant pathogenicity for multiple cancer syndrome genes. Independent data types currently incorporated in the model for assessing BRCA1 and BRCA2 variants include clinically calibrated prior probability of pathogenicity based on variant location and bioinformatic prediction of variant effect, co-segregation, family cancer history profile, co-occurrence with a pathogenic variant in the same gene, breast tumor pathology, and case-control information. Research and clinical data for multifactorial likelihood analysis were collated for 1395 BRCA1/2 predominantly intronic and missense variants, enabling classification based on posterior probability of pathogenicity for 734 variants: 447 variants were classified as (likely) benign, and 94 as (likely) pathogenic; 248 classifications were new or considerably altered relative to ClinVar submissions. Classifications were compared to information not yet included in the likelihood model, and evidence strengths aligned to those recommended for ACMG/AMP classification codes. Altered mRNA splicing or function relative to known non-pathogenic variant controls were moderately to strongly predictive of variant pathogenicity. Variant absence in population datasets provided supporting evidence for variant pathogenicity. These findings have direct relevance for BRCA1 and BRCA2 variant evaluation, and justify the need for gene-specific calibration of evidence types used for variant classification. This article is protected by copyright. All rights reserved.Peer reviewe

Solving patients with rare diseases through programmatic reanalysis of genome-phenome data.

Funder: EC | EC Seventh Framework Programm | FP7 Health (FP7-HEALTH - Specific Programme "Cooperation": Health); doi: https://doi.org/10.13039/100011272; Grant(s): 305444, 305444Funder: Ministerio de Economía y Competitividad (Ministry of Economy and Competitiveness); doi: https://doi.org/10.13039/501100003329Funder: Generalitat de Catalunya (Government of Catalonia); doi: https://doi.org/10.13039/501100002809Funder: EC | European Regional Development Fund (Europski Fond za Regionalni Razvoj); doi: https://doi.org/10.13039/501100008530Funder: Instituto Nacional de Bioinformática ELIXIR Implementation Studies Centro de Excelencia Severo OchoaFunder: EC | EC Seventh Framework Programm | FP7 Health (FP7-HEALTH - Specific Programme "Cooperation": Health)Reanalysis of inconclusive exome/genome sequencing data increases the diagnosis yield of patients with rare diseases. However, the cost and efforts required for reanalysis prevent its routine implementation in research and clinical environments. The Solve-RD project aims to reveal the molecular causes underlying undiagnosed rare diseases. One of the goals is to implement innovative approaches to reanalyse the exomes and genomes from thousands of well-studied undiagnosed cases. The raw genomic data is submitted to Solve-RD through the RD-Connect Genome-Phenome Analysis Platform (GPAP) together with standardised phenotypic and pedigree data. We have developed a programmatic workflow to reanalyse genome-phenome data. It uses the RD-Connect GPAP's Application Programming Interface (API) and relies on the big-data technologies upon which the system is built. We have applied the workflow to prioritise rare known pathogenic variants from 4411 undiagnosed cases. The queries returned an average of 1.45 variants per case, which first were evaluated in bulk by a panel of disease experts and afterwards specifically by the submitter of each case. A total of 120 index cases (21.2% of prioritised cases, 2.7% of all exome/genome-negative samples) have already been solved, with others being under investigation. The implementation of solutions as the one described here provide the technical framework to enable periodic case-level data re-evaluation in clinical settings, as recommended by the American College of Medical Genetics

Solve-RD: systematic pan-European data sharing and collaborative analysis to solve rare diseases.

For the first time in Europe hundreds of rare disease (RD) experts team up to actively share and jointly analyse existing patient's data. Solve-RD is a Horizon 2020-supported EU flagship project bringing together >300 clinicians, scientists, and patient representatives of 51 sites from 15 countries. Solve-RD is built upon a core group of four European Reference Networks (ERNs; ERN-ITHACA, ERN-RND, ERN-Euro NMD, ERN-GENTURIS) which annually see more than 270,000 RD patients with respective pathologies. The main ambition is to solve unsolved rare diseases for which a molecular cause is not yet known. This is achieved through an innovative clinical research environment that introduces novel ways to organise expertise and data. Two major approaches are being pursued (i) massive data re-analysis of >19,000 unsolved rare disease patients and (ii) novel combined -omics approaches. The minimum requirement to be eligible for the analysis activities is an inconclusive exome that can be shared with controlled access. The first preliminary data re-analysis has already diagnosed 255 cases form 8393 exomes/genome datasets. This unprecedented degree of collaboration focused on sharing of data and expertise shall identify many new disease genes and enable diagnosis of many so far undiagnosed patients from all over Europe

Solving unsolved rare neurological diseases-a Solve-RD viewpoint.

Funder: Durch Princess Beatrix Muscle Fund Durch Speeren voor Spieren Muscle FundFunder: University of Tübingen Medical Faculty PATE programFunder: European Reference Network for Rare Neurological Diseases | 739510Funder: European Joint Program on Rare Diseases (EJP-RD COFUND-EJP) | 44140962

Twist exome capture allows for lower average sequence coverage in clinical exome sequencing

Background Exome and genome sequencing are the predominant techniques in the diagnosis and research of genetic disorders. Sufficient, uniform and reproducible/consistent sequence coverage is a main determinant for the sensitivity to detect single-nucleotide (SNVs) and copy number variants (CNVs). Here we compared the ability to obtain comprehensive exome coverage for recent exome capture kits and genome sequencing techniques. Results We compared three different widely used enrichment kits (Agilent SureSelect Human All Exon V5, Agilent SureSelect Human All Exon V7 and Twist Bioscience) as well as short-read and long-read WGS. We show that the Twist exome capture significantly improves complete coverage and coverage uniformity across coding regions compared to other exome capture kits. Twist performance is comparable to that of both short- and long-read whole genome sequencing. Additionally, we show that even at a reduced average coverage of 70× there is only minimal loss in sensitivity for SNV and CNV detection. Conclusion We conclude that exome sequencing with Twist represents a significant improvement and could be performed at lower sequence coverage compared to other exome capture techniques