Effects of Repeated Freeze and Thaw Cycles on the Genome-Wide DNA Methylation Profile of Isolated Genomic DNA.

The characterization of DNA methylation patterns to identify epigenetic markers for complex human diseases is an important and rapidly evolving part in biomedical research. DNA samples collected and stored in clinical biobanks over the past years are an important source for future epigenetic studies. Isolated gDNA is considered stable when stored at low temperatures for several years. However, the effect of multiple use and the associated repeated thawing of long-term stored DNA samples on DNA methylation patterns has not yet been investigated. In this study, we examined the influence of up to 10 freeze and thaw cycles on global DNA methylation by comparing genome-wide methylation profiles. DNA samples from 19 healthy volunteers were either frozen at -80°C or subjected to up to 10 freeze and thaw cycles. Genome-wide DNA methylation was analyzed after 0, 1, 3, 5, or 10 thaw cycles using the Illumina Infinium MethylationEPIC BeadChip. Evaluation of the global DNA methylation profile by beta-value density plots and multidimensional scaling plots revealed an expected clear participant-dependent variability, but a very low variability depending on the freeze and thaw cycles. In accordance, no significant difference in any of the methylated cytosine/guanine sites studied could be detected in the performed statistical analyses. Our results suggest that long-term frozen DNA samples are still suitable for epigenetic studies after multiple thaw cycles.

The Changing Epidemiology of Viral Hepatitis in a Post-Soviet Country-The Case of Kyrgyzstan.

Historically, viral hepatitis has been a considerable public health problem in Central Asian countries, which may have worsened after the dissolution of the Soviet Union. However, up-to-date seroepidemiological studies are lacking. The aim of the present study was, therefore, to provide current estimates of the seroprevalence of viral hepatitis in Kyrgyzstan, one of the economically least developed countries in the region. We conducted a population-based cross-sectional study in 2018 in the capital of Kyrgyzstan, Bishkek (n = 1075). Participants, children and adults, were recruited from an outpatient clinic. The data were collected during face-to-face interviews. A blood sample (6 mL) was collected from each participant and tested with ELISA for the presence of serological markers for five viral hepatitides (A, B, C, D, and E). Post-stratification weighing was performed to obtain nationally representative findings. The overwhelming majority of the study participants were positive for anti-HAV (estimated seroprevalence, 75.3%; 95% confidence interval, 72.5-77.9%). The weighted seroprevalence estimates of HBsAg, anti-HCV, and anti-HDV were 2.2% (1.5-3.3%), 3.8% (2.8-5.1%), and 0.40% (0.15-1.01%), respectively. Anti-HEV seropositivity was 3.3% (2.4-4.5%). Of the 33 HBsAg-positive participants, five (15%) were anti-HDV-positive. Our study confirms that Kyrgyzstan remains a highly endemic country for hepatitis virus A and C infections. However, seroprevalences of HBV and HDV were lower than previously reported, and based on these data, the country could potentially be reclassified from high to (lower) intermediate endemicity. The observed anti-HEV seroprevalence resembles the low endemicity pattern characteristic of high-income countries.

Comparative Magnitude and Persistence of Humoral SARS-CoV-2 Vaccination Responses in the Adult Population in Germany.

Recent increases in SARS-CoV-2 infections have led to questions about duration and quality of vaccine-induced immune protection. While numerous studies have been published on immune responses triggered by vaccination, these often focus on studying the impact of one or two immunisation schemes within subpopulations such as immunocompromised individuals or healthcare workers. To provide information on the duration and quality of vaccine-induced immune responses against SARS-CoV-2, we analyzed antibody titres against various SARS-CoV-2 antigens and ACE2 binding inhibition against SARS-CoV-2 wild-type and variants of concern in samples from a large German population-based seroprevalence study (MuSPAD) who had received all currently available immunisation schemes. We found that homologous mRNA-based or heterologous prime-boost vaccination produced significantly higher antibody responses than vector-based homologous vaccination. Ad26.CoV2S.2 performance was particularly concerning with reduced titres and 91.7% of samples classified as non-responsive for ACE2 binding inhibition, suggesting that recipients require a booster mRNA vaccination. While mRNA vaccination induced a higher ratio of RBD- and S1-targeting antibodies, vector-based vaccines resulted in an increased proportion of S2-targeting antibodies. Given the role of RBD- and S1-specific antibodies in neutralizing SARS-CoV-2, their relative over-representation after mRNA vaccination may explain why these vaccines have increased efficacy compared to vector-based formulations. Previously infected individuals had a robust immune response once vaccinated, regardless of which vaccine they received, which could aid future dose allocation should shortages arise for certain manufacturers. Overall, both titres and ACE2 binding inhibition peaked approximately 28 days post-second vaccination and then decreased.

Children and Adolescents' Behavioral Patterns in Response to Escalating COVID-19 Restriction Reveal Sex and Age Differences.

PURPOSE: The COVID-19 pandemic affects students in a myriad of different ways. Our prospective, longitudinal study in a cohort of students in Hannover, Germany explores behavioral patterns during escalating COVID-19 restrictions. METHODS: In total, 777 students between the age of 9 and 20 were assessed for their activity engagement, travel patterns, and self-assessed compliance with protective recommendations at six time points between June 2020 and June 2021 (3,564 observations) and were monitored for severe acute respiratory syndrome coronavirus 2 infection by nasal swab polymerase chain reaction and serum antibody titers. RESULTS: Activity engagement decreased, but self-assessed compliance with measures such as mask wearing and social distancing was stable during escalating restrictions. Although we found no sex difference during the summer break, when incidence was lowest, females engaged in a higher variety of activities than males for all other time points. Older students engaged in more activities and self-assigned themselves lower compliance values than younger ones. Greater involvement in different activities was seen in households which traveled more frequently. Infection rate in our cohort was low (0.03% acute infections, 1.94% positive seroprevalence). DISCUSSION: Our study supports the view that, overall, students show high compliance with COVID-19 recommendations and restrictions. The identification of subsets, such as female and older students, with higher risk behavioral patterns should be considered when implementing public information campaigns. In light of the low infection rate in our cohort, we conclude that in-person learning can occur safely if extensive protective measures are in place and the incidence in the general population remains moderate.

Plasma Metabolome Signature Indicative of BRCA1 Germline Status Independent of Cancer Incidence.

Individuals carrying a pathogenic germline variant in the breast cancer predisposition gene BRCA1 (gBRCA1+) are prone to developing breast cancer. Apart from its well-known role in DNA repair, BRCA1 has been shown to powerfully impact cellular metabolism. While, in general, metabolic reprogramming was named a hallmark of cancer, disrupted metabolism has also been suggested to drive cancer cell evolution and malignant transformation by critically altering microenvironmental tissue integrity. Systemic metabolic effects induced by germline variants in cancer predisposition genes have been demonstrated before. Whether or not systemic metabolic alterations exist in gBRCA1+ individuals independent of cancer incidence has not been investigated yet. We therefore profiled the plasma metabolome of 72 gBRCA1+ women and 72 age-matched female controls, none of whom (carriers and non-carriers) had a prior cancer diagnosis and all of whom were cancer-free during the follow-up period. We detected one single metabolite, pyruvate, and two metabolite ratios involving pyruvate, lactate, and a metabolite of yet unknown structure, significantly altered between the two cohorts. A machine learning signature of metabolite ratios was able to correctly distinguish between gBRCA1+ and controls in ~82%. The results of this study point to innate systemic metabolic differences in gBRCA1+ women independent of cancer incidence and raise the question as to whether or not constitutional alterations in energy metabolism may be involved in the etiology of BRCA1-associated breast cancer.

Genome-wide association study identifies 48 common genetic variants associated with handedness.

Handedness has been extensively studied because of its relationship with language and the over-representation of left-handers in some neurodevelopmental disorders. Using data from the UK Biobank, 23andMe and the International Handedness Consortium, we conducted a genome-wide association meta-analysis of handedness (N = 1,766,671). We found 41 loci associated (P < 5 × 10-8) with left-handedness and 7 associated with ambidexterity. Tissue-enrichment analysis implicated the CNS in the aetiology of handedness. Pathways including regulation of microtubules and brain morphology were also highlighted. We found suggestive positive genetic correlations between left-handedness and neuropsychiatric traits, including schizophrenia and bipolar disorder. Furthermore, the genetic correlation between left-handedness and ambidexterity is low (rG = 0.26), which implies that these traits are largely influenced by different genetic mechanisms. Our findings suggest that handedness is highly polygenic and that the genetic variants that predispose to left-handedness may underlie part of the association with some psychiatric disorders.

Quality Assessment of the Preanalytical Workflow in Liquid Biobanking: Taurine as a Serum-Specific Quality Indicator for Preanalytical Process Variations.

The scientific impact of translational biomedical research largely depends on the availability of high-quality biomaterials. However, evidence-based and robust quality indicators (QIs) covering the most relevant preanalytical variations are still lacking. The aim of this study was to identify and validate a QI suitable for assessing time-to-centrifugation (TTC) delays in human liquid biospecimens originating from both healthy and diseased individuals. Serum and plasma samples with varying TTCs were analyzed by liquid chromatography coupled to tandem mass spectrometry (LC-MS/MS) in a pilot cohort of healthy individuals to identify a suitable QI candidate. Taurine (TAU), as a TTC QI candidate, was validated in healthy individuals and patients with rheumatologic and cardiologic diseases, considering the (1) preanalytical handling temperature, (2) platelet count, and (3) postcentrifugation delay. For discrimination of high TTC (TTC >60 minutes) from low TTC serum specimens, a probability calculation tool was developed (Triple-T-cutoff-model). TTC-dependent changes in healthy individuals were observed for amino acids, particularly TAU. Validation of the TAU levels in an independent cohort of healthy individuals revealed a time-dependent increase in serum, but not in plasma, for a TTC delay of 30-240 minutes. TAU increases were dependent on the handling temperature and platelet count and volume. By contrast, no changes in TAU concentrations were observed for additional postcentrifugation delays. Validation of TAU and the Triple-T-cutoff-model, in rheumatologic/cardiologic patient collectives, allowed the discrimination of samples with TTC ≤60 min/>60 min with estimated AUROC (area under the receiver operating characteristic curve) values of 89% [78%-100%]/86% [71%-100%] and 91% [79%-100%]/84% [68%-100%], respectively. Considering the preanalytical handling temperature and platelet count and volume, TAU and the Triple-T-cutoff-model represent reliable QIs for TTC >60 minutes in serum samples from healthy individuals and selected rheumatologic/cardiologic patients. However, further studies in larger patient collectives with various diseases are needed to assess the robustness and potential of the QIs presented in this article as biobanking quality assurance/quality control tools to support high-quality biomedical research.

Whole blood microRNA levels associate with glycemic status and correlate with target mRNAs in pathways important to type 2 diabetes.

We analyzed the associations between whole blood microRNA profiles and the indices of glucose metabolism and impaired fasting glucose and examined whether the discovered microRNAs correlate with the expression of their mRNA targets. MicroRNA and gene expression profiling were performed for the Young Finns Study participants (n = 871). Glucose, insulin, and glycated hemoglobin (HbA1c) levels were measured, the insulin resistance index (HOMA2-IR) was calculated, and the glycemic status (normoglycemic [n = 534]/impaired fasting glucose [IFG] [n = 252]/type 2 diabetes [T2D] [n = 24]) determined. Levels of hsa-miR-144-5p, -122-5p, -148a-3p, -589-5p, and hsa-let-7a-5p associated with glycemic status. hsa-miR-144-5p and -148a-3p associated with glucose levels, while hsa-miR-144-5p, -122-5p, -184, and -339-3p associated with insulin levels and HOMA2-IR, and hsa-miR-148a-3p, -15b-3p, -93-3p, -146b-5p, -221-3p, -18a-3p, -642a-5p, and -181-2-3p associated with HbA1c levels. The targets of hsa-miR-146b-5p that correlated with its levels were enriched in inflammatory pathways, and the targets of hsa-miR-221-3p were enriched in insulin signaling and T2D pathways. These pathways showed indications of co-regulation by HbA1c-associated miRNAs. There were significant differences in the microRNA profiles associated with glucose, insulin, or HOMA-IR compared to those associated with HbA1c. The HbA1c-associated miRNAs also correlated with the expression of target mRNAs in pathways important to the development of T2D.

Differentially expressed genes and canonical pathways in the ascending thoracic aortic aneurysm - The Tampere Vascular Study.

Ascending thoracic aortic aneurysm (ATAA) is a multifactorial disease with a strong inflammatory component. Surgery is often required to prevent aortic rupture and dissection. We performed gene expression analysis (Illumina HumanHT-12 version 3 Expression BeadChip) for 32 samples from ATAA (26 without/6 with dissection), and 28 left internal thoracic arteries (controls) collected in Tampere Vascular study. We compared expression profiles and conducted pathway analysis using Ingenuity Pathway Analysis (IPA) to reveal differences between ATAA and a healthy artery wall. Almost 5000 genes were differentially expressed in ATAA samples compared to controls. The most downregulated gene was homeobox (HOX) A5 (fold change, FC = -25.3) and upregulated cadherin-2 (FC = 12.6). Several other HOX genes were also found downregulated (FCs between -25.3 and -1.5, FDR < 0.05). 43, mostly inflammatory, canonical pathways in ATAA were found to be significantly (p < 0.05, FDR < 0.05) differentially expressed. The results remained essentially the same when the 6 dissected ATAA samples were excluded from the analysis. We show for the first time on genome level that ATAA is an inflammatory process, revealing a more detailed molecular pathway level pathogenesis. We propose HOX genes as potentially important players in maintaining aortic integrity, altered expression of which might be important in the pathobiology of ATAA.

Establishment of a cohort for deep phenotyping of the immune response to influenza vaccination among elderly individuals recruited from the general population.

Elderly individuals have the highest burden of disease from influenza infection but also the lowest immune response to influenza vaccination. A better understanding of the host response to influenza vaccination in the elderly is therefore urgently needed. We conducted a biphasic prospective, population-based study from Dec. 2014 to May 2015 (pilot study) and Sept. 2015 to May 2016 (main study). Individuals 65-80 y of age were randomly selected from the residents' registration office in Hannover, Germany, for the pilot (n = 34) and main study (n = 200). The pilot study tested recruitment for study arms featuring 2, 4, or 5 visits/blood draws. The 5-visit (day 0, 1/3, 7, 21, 70 with respect to vaccination) study arm was selected for the main study. Both studies featured vaccination with Fluad™ (Novartis, Italy), a detailed medical history, a physical exam, recording of adverse events, completion of a questionnaire on common infections and an end-of-study questionnaire, and blood samples. Response rates in the pilot and main studies were 3.7% and 4.0%, respectively. Willingness to participate did not differ among the study arms (Fisher's exact test, p = 0.44). In both studies, there were no losses to follow-up. Compliance with study visits, blood sampling and completion of the questionnaires was very high (100%, >97%, 100%, respectively), as were participants' acceptance of and satisfaction with both phases of the study. The low response rates indicate the need for optimized recruitment strategies if the study population is to be representative of the general population. Nonetheless, the complex prospective study design proved to be highly feasible.

Motivations for (non)participation in population-based health studies among the elderly - comparison of participants and nonparticipants of a prospective study on influenza vaccination.

BACKGROUND: Participation in epidemiological studies has strongly declined in recent years. We examined the reasons for (non)participation in population-based health studies among participants and nonparticipants of a prospective study on influenza vaccination among the elderly. METHODS: Males and females between 65 and 80 years of age (N = 5582) were randomly selected from the residents' registration office in Hannover, Germany, and were invited to participate in a study featuring vaccination with a seasonal adjuvanted influenza vaccine (Fluad™, Novartis) including five follow-up visits (day 0, 1/3, 7, 21, 70 with respect to vaccination). A 24-item nonresponder questionnaire, including 10 items on reasons for participating in a hypothetical health study, was mailed to 1500 randomly selected nonparticipants. The same 10 items were included in the end-of-study questionnaire administered to the participants in the vaccination study (n = 200). Logistic regression analysis with backward elimination was used to identify the reasons most strongly associated with nonparticipation. RESULTS: Five hundred thirty-one (35%) nonparticipants and 200 participants (100%) returned the respective questionnaires. Nonparticipation was associated with a lower interest in obtaining personal health information (OR = 3.32) and a preference for less invasive (OR = 3.01) and less time-demanding (OR = 2.19) studies. Responses to other items, e.g. regarding altruistic motives, monetary compensation, general interest of the study, or study approval through ethics committee and data security authority, did not differ between participants and nonparticipants. CONCLUSIONS: Participation rates in health studies among elderly individuals could potentially be improved by reducing interventions and time demand, for instance by implementing methods of self-sampling and remote data collection. TRIAL REGISTRATION: No. 1100359 (ClinicalTrials.gov, date of registration: 09.02.2015).

Differentially expressed genes and canonical pathway expression in human atherosclerotic plaques - Tampere Vascular Study.

Cardiovascular diseases due to atherosclerosis are the leading cause of death globally. We aimed to investigate the potentially altered gene and pathway expression in advanced peripheral atherosclerotic plaques in comparison to healthy control arteries. Gene expression analysis was performed (Illumina HumanHT-12 version 3 Expression BeadChip) for 68 advanced atherosclerotic plaques (15 aortic, 29 carotid and 24 femoral plaques) and 28 controls (left internal thoracic artery (LITA)) from Tampere Vascular Study. Dysregulation of individual genes was compared to healthy controls and between plaques from different arterial beds and Ingenuity pathway analysis was conducted on genes with a fold change (FC) > ±1.5 and false discovery rate (FDR) < 0.05. 787 genes were significantly differentially expressed in atherosclerotic plaques. The most up-regulated genes were osteopontin and multiple MMPs, and the most down-regulated were cell death-inducing DFFA-like effector C and A (CIDEC, CIDEA) and apolipoprotein D (FC > 20). 156 pathways were differentially expressed in atherosclerotic plaques, mostly inflammation-related, especially related with leukocyte trafficking and signaling. In artery specific plaque analysis 50.4% of canonical pathways and 41.2% GO terms differentially expressed were in common for all three arterial beds. Our results confirm the inflammatory nature of advanced atherosclerosis and show novel pathway differences between different arterial beds.

Blood hsa-miR-122-5p and hsa-miR-885-5p levels associate with fatty liver and related lipoprotein metabolism-The Young Finns Study.

MicroRNAs are involved in disease development and may be utilized as biomarkers. We investigated the association of blood miRNA levels and a) fatty liver (FL), b) lipoprotein and lipid pathways involved in liver lipid accumulation and c) levels of predicted mRNA targets in general population based cohort. Blood microRNA profiling (TaqMan OpenArray), genome-wide gene expression arrays and nuclear magnetic resonance metabolomics were performed for Young Finns Study participants aged 34-49 years (n = 871). Liver fat status was assessed ultrasonographically. Levels of hsa-miR-122-5p and -885-5p were up-regulated in individuals with FL (fold change (FC) = 1.55, p = 1.36 * 10-14 and FC = 1.25, p = 4.86 * 10-4, respectively). In regression model adjusted with age, sex and BMI, hsa-miR-122-5p and -885-5p predicted FL (OR = 2.07, p = 1.29 * 10-8 and OR = 1.41, p = 0.002, respectively). Together hsa-miR-122-5p and -885-5p slightly improved the detection of FL beyond established risk factors. These miRNAs may be associated with FL formation through the regulation of lipoprotein metabolism as hsa-miR-122-5p levels associated with small VLDL, IDL, and large LDL lipoprotein subclass components, while hsa-miR-885-5p levels associated inversely with XL HDL cholesterol levels. Hsa-miR-885-5p levels correlated inversely with oxysterol-binding protein 2 (OSBPL2) expression (r = -0.143, p = 1.00 * 10-4) and suppressing the expression of this lipid receptor and sterol transporter could link hsa-miR-885-5p with HDL cholesterol levels.

Talin and vinculin are downregulated in atherosclerotic plaque; Tampere Vascular Study.

BACKGROUND AND AIMS: Focal adhesions (FA) play an important role in the tissue remodeling and in the maintenance of tissue integrity and homeostasis. Talin and vinculin proteins are among the major constituents of FAs contributing to cellular well-being and intercellular communication. METHODS: Microarray analysis (MA) and qRT-PCR low-density array were implemented to analyze talin-1, talin-2, meta-vinculin and vinculin gene expression in circulating blood and arterial plaque. RESULTS: All analyzed genes were significantly and consistently downregulated in plaques (carotid, abdominal aortic and femoral regions) compared to left internal thoracic artery (LITA) control. The use of LITA samples as controls for arterial plaque samples was validated using immunohistochemistry by comparing LITA samples with healthy arterial samples from a cadaver. Even though the differences in expression levels between stable and unstable plaques were not statistically significant, we observed further negative tendency in the expression in unstable atherosclerotic plaques. The confocal tissue imaging revealed gradient of talin-1 expression in plaque with reduction close to the vessel lumen. Similar gradient was observed for talin-2 expression in LITA controls but was not detected in plaques. This suggests that impaired tissue mechanostability affects the tissue remodeling and healing capabilities leading to development of unstable plaques. CONCLUSIONS: The central role of talin and vinculin in cell adhesions suggests that the disintegration of the tissue in atherosclerosis could be partially driven by downregulation of these genes, leading to loosening of cell-ECM interactions and remodeling of the tissue.

[Centralized biobanks: a basis for medical research]. / Zentralisierte Biobanken als Grundlage für die medizinische Forschung.

Biobanks are the basis for a substantial part of biomedical research. The development, establishment and operation of biobanks are connected to a broad range of aspects, mainly concerning the preparation, storage, usage and dissemination of samples and associated data, in addition to the social and public involvement of these processes. These complex requirements can often only be managed in large centralized biobanks. In recent years, centralized clinical biobanks have been established in several university clinics in Germany. Similar activities take place in other European countries and worldwide. This article highlights the requirements and main tasks of centralized clinical biobanks: high-quality pre-analytics and sample storage, the creation of professional IT structures, data protection, ethical issues, in addition to quality and project management.

Kindlin 3 (FERMT3) is associated with unstable atherosclerotic plaques, anti-inflammatory type II macrophages and upregulation of beta-2 integrins in all major arterial beds.

BACKGROUND: Kindlins (FERMT) are cytoplasmic proteins required for integrin (ITG) activation, leukocyte transmigration, platelet aggregation and thrombosis. Characterization of kindlins and their association with atherosclerotic plaques in human(s) is lacking. METHODS AND RESULTS: Exploratory microarray (MA) was first performed followed by selective quantitative validation of robustly expressed genes with qRT-PCR low-density array (LDA). In LDA, ITGA1 (1.30-fold, p = 0.041) and ITGB3 (1.37-fold, p = 0.036) were upregulated in whole blood samples of patients with coronary artery disease (CAD) compared to healthy controls. In arterial plaques, both robustly expressed transcript variants of FERMT3 (MA: 5.90- and 3.4-fold; LDA: 3.99-fold, p < 0.0001 for all) and ITGB2 (MA: 4.81- and 4.92-fold; LDA: 5.29-fold, p < 0.0001 for all) were upregulated while FERMT2 was downregulated (MA: -1.61-fold; LDA: -2.88-fold, p < 0.0001 for both). The other integrins (ITGA1, ITGAV, ITGB3, ITGB5) were downregulated. All these results were replicated in at least one arterial bed. The latter FERMT3 transcript variant associated with unstable plaques (p = 0.0004). FERMT3 correlated with M2 macrophage markers and in hierarchical cluster analysis clustered with inflammatory and macrophage markers, while FERMT2 correlated with SMC-rich plaque markers and clustered with SMC markers. In confocal immunofluorescence analysis, FERMT3 protein colocalized with abundant CD68-positive cells of monocytic origin in the atherosclerotic plaques, while co-localization of FERMT3 with HHF35 indicative of smooth muscle cells was low. CONCLUSIONS: Kindlin-3 (FERMT3) is upregulated in atherosclerotic, especially unstable plaques, mainly in cells of monocytic origin and of M2 type. Simultaneous upregulation of ITGB2 suggests a synergistic effect on leukocyte adherence and transmigration into the vessel wall.

Polymorphisms related to ORMDL3 are associated with asthma susceptibility, alterations in transcriptional regulation of ORMDL3, and changes in TH2 cytokine levels.

BACKGROUND: Chromosome 17q21, harboring the orosomucoid 1-like 3 (ORMDL3) gene, has been consistently associated with childhood asthma in genome-wide association studies. OBJECTIVE: We investigated genetic variants in and around ORMDL3 that can change the function of ORMDL3 and thus contribute to asthma susceptibility. METHODS: We performed haplotype analyses and fine mapping of the ORMDL3 locus in a cross-sectional (International Study of Asthma and Allergies in Childhood Phase II, n = 3557 total subjects, n = 281 asthmatic patients) and case-control (Multicenter Asthma Genetics in Childhood Study/International Study of Asthma and Allergies in Childhood Phase II, n = 1446 total subjects, n = 763 asthmatic patients) data set to identify putative causal single nucleotide polymorphisms (SNPs) in the locus. Top asthma-associated polymorphisms were analyzed for allele-specific effects on transcription factor binding and promoter activity in vitro and gene expression in PBMCs after stimulation ex vivo. RESULTS: Two haplotypes (H1 and H2) were significantly associated with asthma in the cross-sectional (P = 9.9 × 10(-5) and P = .0035, respectively) and case-control (P = 3.15 × 10(-8) and P = .0021, respectively) populations. Polymorphisms rs8076131 and rs4065275 were identified to drive these effects. For rs4065275, a quantitative difference in transcription factor binding was found, whereas for rs8076131, changes in upstream stimulatory factor 1 and 2 transcription factor binding were observed in vitro by using different cell lines and PBMCs. This might contribute to detected alterations in luciferase activity paralleled with changes in ORMDL3 gene expression and IL-4 and IL-13 cytokine levels ex vivo in response to innate and adaptive stimuli in an allele-specific manner. Both SNPs were in strong linkage disequilibrium with asthma-associated 17q21 SNPs previously related to altered ORMDL3 gene expression. CONCLUSION: Polymorphisms in a putative promoter region of ORMDL3, which are associated with childhood asthma, alter transcriptional regulation of ORMDL3, correlate with changes in TH2 cytokines levels, and therefore might contribute to the childhood asthma susceptibility signal from 17q21.

Rationale, design and objectives of ARegPKD, a European ARPKD registry study.

BACKGROUND: Autosomal recessive polycystic kidney disease (ARPKD) is a rare but frequently severe disorder that is typically characterized by cystic kidneys and congenital hepatic fibrosis but displays pronounced phenotypic heterogeneity. ARPKD is among the most important causes for pediatric end stage renal disease and a leading reason for liver-, kidney- or combined liver kidney transplantation in childhood. The underlying pathophysiology, the mechanisms resulting in the observed clinical heterogeneity and the long-term clinical evolution of patients remain poorly understood. Current treatment approaches continue to be largely symptomatic and opinion-based even in most-advanced medical centers. While large clinical trials for the frequent and mostly adult onset autosomal dominant polycystic kidney diseases have recently been conducted, therapeutic initiatives for ARPKD are facing the challenge of small and clinically variable cohorts for which reliable end points are hard to establish. METHODS/DESIGN: ARegPKD is an international, mostly European, observational study to deeply phenotype ARPKD patients in a pro- and retrospective fashion. This registry study is conducted with the support of the German Society for Pediatric Nephrology (GPN) and the European Study Consortium for Chronic Kidney Disorders Affecting Pediatric Patients (ESCAPE Network). ARegPKD clinically characterizes long-term ARPKD courses by a web-based approach that uses detailed basic data questionnaires in combination with yearly follow-up visits. Clinical data collection is accompanied by associated biobanking and reference histology, thus setting roots for future translational research. DISCUSSION: The novel registry study ARegPKD aims to characterize miscellaneous subcohorts and to compare the applied treatment options in a large cohort of deeply characterized patients. ARegPKD will thus provide evidence base for clinical treatment decisions and contribute to the pathophysiological understanding of this severe inherited disorder.

Predicting sudden cardiac death using common genetic risk variants for coronary artery disease.

AIMS: Genome-wide association studies (GWAS) have identified many variants associating with an increased risk of coronary artery disease (CAD). We studied the possible association between these variants and the risk of sudden cardiac death (SCD). METHODS AND RESULTS: A weighted genetic risk score (GRSCAD) was formed from variants most strongly associating with CAD identified by the CARDIoGRAMplusC4D Consortium explaining 10.6% of the heritability of CAD [153 single-nucleotide polymorphisms with r(2) < 0.2]. The association between GRSCAD and the occurrence of SCD was studied in three independent autopsy series of consecutive cases combining altogether 1035 autopsies with 306 SCDs due to CAD (SCDCAD). The results were replicated in a prospective follow-up study of 2321 patients (mean follow-up time of 6.2 years with 48 incident SCDs of which 39 due to CAD) undergoing clinical exercise test at baseline. In a meta-analysis of the autopsy series, GRSCAD associated significantly with the risk of SCDCAD with age, body mass index, and sex adjusted odds ratio (OR) of 1.042 (1.023-1.061, P = 9.1 × 10(-6)) for one allele increase in GRSCAD. The same association was seen in both sexes. GRSCAD predicted significantly the risk of SCDCAD also in a prospective study setting (Cox regression analysis adjusted with all relevant clinical data): hazard ratio 1.049 (1.010-1.090, P = 0.014). In meta-analysis of all cohorts (adjusting further for other genetic markers related to traditional risk factors and QT-interval), the association was highly significant [OR 1.045 (1.028-1.063), P = 1.7 × 10(-7)]. CONCLUSION: Genetic risk estimate for CAD may also be used to predict SCD.

A genome-wide expression quantitative trait loci analysis of proprotein convertase subtilisin/kexin enzymes identifies a novel regulatory gene variant for FURIN expression and blood pressure.

Proprotein convertase subtilisin/kexin (PCSK) enzymes cleave and convert their immature substrates into biologically active forms. Polymorphisms in the PCSK genes have been reported to associate with human diseases and phenotypes, including hypercholesterolemia and blood pressure (BP), and targeting PCSKs is considered a promising future form of drug therapy. PCSK processing is readily induced upon upregulation of the enzyme, but the genetic factors contributing to PCSK expression have not been thoroughly characterized. To gain a comprehensive understanding of the genetic regulation of PCSK expression, we performed, for the first time, a genome-wide expression quantitative trait loci (eQTL) analysis using mRNA expression in >1400 human peripheral blood samples from the Cardiovascular Risk in Young Finns Study and ca. ten million single-nucleotide polymorphisms (SNPs). The expression data showed clear expression for FURIN, PCSK5, PCSK7 and MBTPS1 (membrane-bound transcription factor peptidase, site 1) mRNAs in virtually all tested samples. A discovery analysis demonstrated a genome-wide significant (p < 5 × 10(-8)) association with the selected PCSK probes for 1024 variants, which were located at ten independent loci. Of these loci, 5/10 could be confirmed to regulate PCSK expression in two additional and independent sample sets. Finally, a phenotypic analysis demonstrated that a novel cis-eQTL SNP rs4702 for FURIN is strongly associated with both diastolic (p = 0.012) and systolic (p = 0.035) BP levels, as well as peripheral vascular resistance (p = 0.003). These findings indicate that the expression of the PCSK enzymes is regulated by genetic factors, which have biological roles in health and disease.