The single nucleotide polymorphism rs4986790 (c.896A>G) in the gene TLR4 as a protective factor in corona virus disease 2019 (COVID-19).

Background and aims: Several factors, such as hypertension and diabetes mellitus, are known to influence the course of coronavirus disease 2019 (COVID-19). However, there is currently little information on genetic markers that influence the severity of COVID-19. In this study, we specifically investigated the single nucleotide polymorphism (SNP) rs4986790 in the TLR4 gene to identify a universal marker for preclinical prediction of COVID-19 disease progression. Methods: We analyzed the influence of demographics, pre-existing conditions, inflammatory parameters at the time of hospitalization, and TLR4 rs4986790 genotype on the outcome of COVID-19 in a comprehensive cohort (N = 1570). We performed multivariable analysis to investigate the impact of each factor. Results: We confirmed that younger patient age and absence of pre-existing conditions were protective factors against disease progression. Furthermore, when comparing patients with mild SARS-CoV-2 infection with patients who required hospitalization or intensive care or even died due to COVID-19, the AG/GG genotype of TLR4 rs4986790 was found to be a protective factor against COVID-19 disease progression (OR: 0.51, 95% CI: 0.34 - 0.77, p = 0.001). In addition, we demonstrated that low levels of interleukin-6 (IL-6) and procalcitonin (PCT) had a favorable effect on COVID-19 disease severity. In the subsequent multivariable analysis, we confirmed the absence of cardiovascular disease, low levels of IL-6 and PCT, and TLR4 rs4986790 AG/GG genotypes as independent predictors of potential hospitalization and reduction of severe or fatal disease course. Conclusion: In this study, we identified an additional genetic factor that may serve as an invariant predictor of COVID-19 outcome. The TLR4 rs4986790 AG/GG genotype reduced by half the risk of COVID-19 patients requiring hospitalization, intensive care or to have a fatal outcome. In addition, we were able to confirm the influence of previously known factors such as pre-existing conditions and inflammatory markers upon the onset of disease on the course of COVID-19. Based on these observations, we hereby provide another prognostic biomarker that could be used in routine diagnostics as a predictive factor for the severity of COVID-19 prior to SARS-CoV-2 infection.

Single-Nucleotide Polymorphism in Genes Encoding G Protein Subunits GNB3 and GNAQ Increase the Risk of Cardiovascular Morbidity among Patients Undergoing Renal Replacement Therapy.

Single-nucleotide polymorphisms in G protein subunits are linked to an increased risk of cardiovascular events among the general population. We assessed the effects of GNB3 c.825C > T, GNAQ -695/-694GC > TT, and GNAS c.393C > T polymorphisms on the risk of cardiovascular events among 454 patients undergoing renal replacement therapy. The patients were followed up for a median of 4.5 years after the initiation of dialysis. Carriers of the TT/TT genotype of GNAQ required stenting because of coronary artery stenosis (p = 0.0009) and developed cardiovascular events involving more than one organ system (p = 0.03) significantly earlier and more frequently than did the GC/TT or GC/GC genotypes. Multivariate analysis found that the TT/TT genotype of GNAQ was an independent risk factor for coronary artery stenosis requiring stent (hazard ratio, 4.5; p = 0.001), cardiovascular events (hazard ratio, 1.93; p = 0.04) and cardiovascular events affecting multiple organs (hazard ratio, 4.9; p = 0.03). In the subgroup of male patients left ventricular dilatation with abnormally increased LVEDD values occurred significantly more frequently in TT genotypes of GNB3 than in CT/CC genotypes (p = 0.007). Our findings suggest that male dialysis patients carrying the TT genotype of GNB3 are at higher risk of left ventricular dilatation and that dialysis patients carrying the TT/TT genotype of GNAQ are prone to coronary artery stenosis and severe cardiovascular events.

Influence of the Single Nucleotide Polymorphisms rs12252 and rs34481144 in IFITM3 on the Antibody Response after Vaccination against COVID-19.

The COVID-19 mRNA vaccine is the first mRNA vaccine approved for human administration by both the U.S. Food and Drug Administration and the European Medicines Agency. Studies have shown that the immune response and the decay of immunity after vaccination with the COVID-19 vaccines are variable within a population. Host genetic factors probably contribute to this variability. In this study, we investigated the effect of the single-nucleotide polymorphisms rs12252 and rs34481144 in the interferon-induced transmembrane protein (IFITM) 3 gene on the humoral immune response after vaccination against COVID-19 with mRNA vaccines. Blood samples were collected from 1893 healthcare workers and medical students at multiple time points post-vaccination and antibody titers against the SARS-CoV-2 S1 protein receptor binding domain were determined at all time points. All participants were genotyped for the rs34481144 and rs12252 polymorphisms in the IFITM3 gene. After the second and third vaccinations, antibody titer levels increased at one month and decreased at six months (p < 0.0001) and were higher after the booster vaccination than after the basic immunization (p < 0.0001). Participants vaccinated with mRNA-1273 had a higher humoral immune response than participants vaccinated with BNT162b2. rs12252 had no effect on the antibody response. In contrast, carriers of the GG genotype in rs34481144 vaccinated with BNT162b2 had a lower humoral immune response compared to A allele carriers, which reached statistical significance on the day of the second vaccination (p = 0.03) and one month after the second vaccination (p = 0.04). Further studies on the influence of rs12252 and rs34481144 on the humoral immune response after vaccination against COVID-19 are needed.

Characteristics and Long-Term Outcome of 535 Patients with Autoimmune Hepatitis-The 20-Year Experience of a High-Volume Tertiary Center.

Background and aims: Autoimmune hepatitis (AIH) is a complex and progressive inflammatory liver disease characterized by immune-mediated destruction of the liver parenchyma, hypergammaglobulinemia, the presence of circulating autoantibodies, and good response to immunosuppressive therapy. Since the prevalence of AIH is relatively rare, data on the clinical course and the long-term outcome are scarce. Patients and methods: We retrospectively analyzed the data of 535 well-documented AIH patients treated at the University Hospital Essen between 2000 and 2020. Results: The majority of patients were middle-aged females (75% women, mean age 45 years) with AIH type 1 (97%). Approximately 32% of patients were diagnosed with cirrhosis due to AIH, 29% had concomitant autoimmune (predominantly autoimmune thyroiditis), and 10% had psychiatric diseases, respectively. Skin tumors were the most common malignant diseases (47% of all tumors), while hepatocellular carcinoma rarely occurred (only six cases). Overall long-term mortality and liver-associated mortality were 9.16% and 4.67%, respectively. However, long-term survival was strongly associated with disease remission. Conclusions: Although AIH is a silent disease and cirrhosis is present in many cases, a favorable long-term prognosis can be achieved by consequent immunosuppressive therapy. The incidence of (liver-associated) complications seems to be lower in comparison to other etiologies, such as viral hepatitis or NASH, and mainly depends on the long-term side effects of immunosuppressive therapy.

CC Genotype of GNAS c.393C>T (rs7121) Polymorphism Has a Protective Effect against Development of BK Viremia and BKV-Associated Nephropathy after Renal Transplant

The GNAS gene encodes the alpha-subunit of the stimulatory G-protein (Gαs) in humans and mice. The single-nucleotide polymorphism of GNAS, c.393C>T, is associated with an elevated production of Gαs and an increased formation of cyclic adenosine monophosphate (cAMP). In the present study, we analyzed the effect of this GNAS polymorphism on a renal allograft outcome. We screened a cohort of 436 renal allograft recipients, who were retrospectively followed up for up to 5 years after transplant. GNAS genotypes were determined with polymerase chain reaction restriction fragment length polymorphism (PCR-RFLP) assays. The 393T allele was detected in 319 (73%) recipients (113 recipients with TT and 206 with CT genotype) and the CC genotype in 117 (27%). The CC genotype was associated with a significantly lower frequency of BK viremia (CC, 17 recipients (15%); T 84 (26%)); p = 0.01; TT, 27 vs. CC, 17, p = 0.07; TT, 27 vs. CT, 57, p = 0. 46; CT, 57 vs. CC, 17, p = 0.01) and BKV-associated nephropathy (CC, 3 recipients (3%); T, 27 (8%); p = 0.03; TT,10 vs. CC, 3, p = 0.04; TT, 10 vs. CT,17, p = 0.85; CT, 17 vs. CC,3, p = 0.04) after transplant. BKV-associated nephropathy-free survival was significantly better among CC genotype carriers than among T allele carriers (p = 0.043; TT vs. CC, p = 0.03; CT vs. CC, p = 0.04; TT vs. CT, p = 0.83). Multivariate analysis indicated an independent protective effect of the CC genotype against the development of both BK viremia (relative risk. 0.54; p = 0.04) and BKV-associated nephropathy after renal transplant (relative risk. 0.27; p = 0.036). The GNAS 393 CC genotype seems to protect renal allograft recipients against the development of BK viremia and BKV-associated nephropathy.

GNB3 c.825c>T polymorphism influences T-cell but not antibody response following vaccination with the mRNA-1273 vaccine.

Background: Immune responses following vaccination against COVID-19 with different vaccines and the waning of immunity vary within the population. Genetic host factors are likely to contribute to this variability. However, to the best of our knowledge, no study on G protein polymorphisms and vaccination responses against COVID-19 has been published so far. Methods: Antibodies against the SARS-CoV-2 spike protein and T-cell responses against a peptide pool of SARS-CoV-2 S1 proteins were measured 1 and 6 months after the second vaccination with mRNA-1273 in the main study group of 204 participants. Additionally, antibodies against the SARS-CoV-2 spike protein were measured in a group of 597 participants 1 month after the second vaccination with mRNA-1273. Genotypes of GNB3 c.825C>T were determined in all participants. Results: The median antibody titer against the SARS-CoV-2 spike protein and median values of spots increment in the SARS-CoV-2 IFN-γ ELISpot assay against the S1-peptide pool were significantly decreased from months 1 to 6 (p < 0.0001). Genotypes of GNB3 c.825C>T had no influence on the humoral immune response. At month 1, CC genotype carriers had significantly increased T-cell responses compared to CT (p = 0.005) or TT (p = 0.02) genotypes. CC genotype carriers had an almost 6-fold increased probability compared to TT genotype carriers and an almost 3-fold increased probability compared to T-allele carriers to mount a SARS-CoV-2-specific T-cell response above the median value. Conclusion: CC genotype carriers of the GNB3 c.825C>T polymorphism have an increased T-cell immune response to SARS-CoV-2, which may indicate better T-cell-mediated protection against COVID-19 after vaccination with mRNA-1273.

GNB3 c.825C>T (rs5443) Polymorphism and Risk of Acute Cardiovascular Events after Renal Allograft Transplant

The c.825C>T single-nucleotide polymorphism (rs5443) of the guanine nucleotide-binding protein subunit ß3 (GNB3) results in increased intracellular signal transduction via G-proteins. The present study investigated the effect of the GNB3 c.825C>T polymorphism on cardiovascular events among renal allograft recipients posttransplant. Our retrospective study involved 436 renal allograft recipients who were followed up for up to 8 years after transplant. The GNB3 c.825C>T polymorphism was detected with restriction fragment length polymorphism (RFLP) polymerase chain reaction (PCR). The GNB3 TT genotype was detected in 43 (10%) of 436 recipients. Death due to an acute cardiovascular event occurred more frequently among recipients with the TT genotype (4 [9%]) than among those with the CC/CT genotypes (7 [2%]; p = 0.003). The rates of myocardial infarction (MI)−free survival (p = 0.003) and acute peripheral artery occlusive disease (PAOD)−free survival (p = 0.004) were significantly lower among T-homozygous patients. A multivariate analysis showed that homozygous GNB3 c.825C>T polymorphism exerted only a mild effect for the occurrence of myocardial infarction (relative risk, 2.2; p = 0.065) or acute PAOD (relative risk, 2.4; p = 0.05) after renal transplant. Our results suggest that the homozygous GNB3 T allele exerts noticeable effects on the risk of MI and acute PAOD only in the presence of additional nonheritable risk factors.

The GNB3 c.825C>T (rs5443) polymorphism and protection against fatal outcome of corona virus disease 2019 (COVID-19).

Background and aims: Albeit several factors which influence the outcome of corona virus disease (COVID-19) are already known, genetic markers which may predict the outcome of the disease in hospitalized patients are still very sparse. Thus, in this study, we aimed to analyze whether the single-nucleotide polymorphism (SNP) rs5443 in the gene GNB3, which was associated with higher T cell responses in previous studies, might be a suitable biomarker to predict T cell responses and the outcome of COVID-19 in a comprehensive German cohort. Methods: We analyzed the influence of demographics, pre-existing disorders, laboratory parameters at the time of hospitalization, and GNB3 rs5443 genotype in a comprehensive cohort (N = 1570) on the outcome of COVID-19. In a sub cohort, we analyzed SARS-CoV-2-specific T cell responses and associated GNB3 rs5443 genotypes. We investigated the influence of all factors on COVID-19 fatality in multivariable analysis. Results: We found a younger patient age, normotension or absence of diabetes mellitus or cardiovascular diseases, normal blood cell counts, and low inflammatory markers at hospital admission were protective factors against fatal course of disease. In addition, the rs5443 TT genotype was significantly associated with protection against COVID-19 fatality (OR: 0.60, 95% CI: 0.40-0.92, p = 0.02). We also observed significantly increased SARS-CoV-2-specific T cell responses in rs5443 TT genotype carriers (p = 0.01). Although we observed a significant association of the factors described previously in univariate analysis, only a younger age of the patients, normal blood cell counts, and the GNB3 rs5443 TT genotype remained independent predictors against COVID-19 fatality in multivariable analysis. Conclusion: Immutable predictors for COVID-19 fatality are relatively rare. In this study we could show that the TT genotype of the SNP rs5443 in the gene GNB3 is associated with protection against COVID-19 fatality. It was as well correlated to higher SARS-CoV-2-specific T cell responses, which could result in a milder course of disease in those patients. Based on those observations we hereby provide a further prognostic biomarker, which might be used in routine diagnostics as a predictive factor for COVID-19 mortality already upon hospitalization.

Individuals With Weaker Antibody Responses After Booster Immunization Are Prone to Omicron Breakthrough Infections.

Background: Despite the high level of protection against severe COVID-19 provided by the currently available vaccines some breakthrough infections occur. Until now, there is no information whether a potential risk of a breakthrough infection can be inferred from the level of antibodies after booster vaccination. Methods: Levels of binding antibodies and neutralization capacity after the first, one and six month after the second, and one month after the third (booster) vaccination against COVID-19 were measured in serum samples from 1391 healthcare workers at the University Hospital Essen. Demographics, vaccination scheme, pre-infection antibody titers and neutralization capacity were compared between individuals with and without breakthrough infections. Results: The risk of developing an Omicron breakthrough infection was independent of vaccination scheme, sex, body mass index, smoking status or pre-existing conditions. In participants with low pre-infection anti-spike antibodies (≤ 2641.0 BAU/ml) and weaker neutralization capacity (≤ 65.9%) against Omicron one month after the booster vaccination the risk for developing an Omicron infection was 10-fold increased (P = 0.001; 95% confidence interval, 2.36 - 47.55). Conclusion: Routine testing of anti-SARS-CoV-2 IgG antibodies and surrogate virus neutralization can quantify vaccine-induced humoral immune response and may help to identify subjects who are at risk for a breakthrough infection. The establishment of thresholds for SARS-CoV-2 IgG antibody levels identifying "non"-, "low" and "high"-responders may be used as an indication for re-vaccination.

Development of De Novo Donor-specific HLA Antibodies and AMR in Renal Transplant Patients Depends on CYP3A5 Genotype.

BACKGROUND: The single-nucleotide polymorphism CYP3A5 rs776746 is related to a reduction in the metabolizing activity of the CYP3A5 enzyme. People carrying at least one copy of the wild-type allele, defined as CYP3A5 expressers, exhibit higher clearance and lower trough concentrations of tacrolimus than homozygous nonexpressers, and this difference may affect alloimmunization and allograft function. METHODS: We retrospectively studied 400 kidney transplant recipients treated with a tacrolimus-based immunosuppression regimen to detect CYP3A5 genotype, de novo formation of HLA antibodies and donor-specific antibodies (DSAs), and clinical outcome up to 5 y after transplant. RESULTS: We found that 69 (17%) of the 400 patients were CYP3A5 expressers. During the first 3 y after transplant, CYP3A5 expressers tended to have lower tacrolimus trough levels than nonexpressers, although their tacrolimus dosage was as much as 80% higher. De novo DSAs were found more frequently in CYP3A5 expressers than in nonexpressers (13/69 [19%] versus 33/331 [10%], P = 0.02). De novo DSA-free survival rates (P = 0.02) were significantly lower for expressers than for nonexpressers. CYP3A5 genotype had no effect on allograft failure, but CYP3A5 expressers exhibited a significantly higher frequency of antibody-mediated rejection. CYP3A5 expresser status was an independent risk factor for the development of de novo DSAs (relative risk, 2.34, P = 0.01). CONCLUSIONS: Early detection of CYP3A5 expressers, enabling genotype-based dose adjustment of tacrolimus immediately after renal transplant, may be a useful strategy for reducing the risk of de novo DSA production and antibody-mediated rejection.

Transmembrane serine protease 2 Polymorphisms and Susceptibility to Severe Acute Respiratory Syndrome Coronavirus Type 2 Infection: A German Case-Control Study.

The transmembrane serine protease 2 (TMPRSS2) is the major host protease that enables entry of the severe acute respiratory syndrome coronavirus type 2 (SARS-CoV-2) into host cells by spike (S) protein priming. Single nucleotide polymorphisms (SNPs) in the gene TMPRSS2 have been associated with susceptibility to and severity of H1N1 or H1N9 influenza A virus infections. Functional variants may influence SARS-CoV-2 infection risk and severity of Coronavirus disease 2019 (COVID-19) as well. Therefore, we analyzed the role of SNPs in the gene TMPRSS2 in a German case-control study. We performed genotyping of the SNPs rs2070788, rs383510, and rs12329760 in the gene TMPRSS2 in 239 SARS-CoV-2-positive and 253 SARS-CoV-2-negative patients. We analyzed the association of the SNPs with susceptibility to SARS-CoV-2 infection and severity of COVID-19. SARS-CoV-2-positive and SARS-CoV-2-negative patients did not differ regarding their demographics. The CC genotype of TMPRSS2 rs383510 was associated with a 1.73-fold increased SARS-CoV-2 infection risk, but was not correlated to severity of COVID-19. Neither TMPRSS2 rs2070788 nor rs12329760 polymorphisms were related to SARS-CoV-2 infection risk or severity of COVID-19. In a multivariable analysis (MVA), the rs383510 CC genotype remained an independent predictor for a 2-fold increased SARS-CoV-2 infection risk. In summary, our report appears to be the first showing that the intron variant rs383510 in the gene TMPRSS2 is associated with an increased risk to SARS-CoV-2 infection in a German cohort.

ACE2 polymorphism and susceptibility for SARS-CoV-2 infection and severity of COVID-19.

OBJECTIVES: The RNA virus severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is responsible for coronavirus disease 2019 (COVID-19). Cell entry is mediated by the human angiotensin-converting enzyme II (ACE2). ACE2 and its close homolog angiotensin-converting enzyme I (ACE) are currently discussed candidate genes, in which single-nucleotide polymorphisms (SNPs) could alter binding or entry of SARS-CoV-2 and enhance tissue damage in the lung or other organs. This could increase the susceptibility for SARS-CoV-2 infection and the severity of COVID-19. PATIENTS AND METHODS: We performed genotyping of SNPs in the genes ACE2 and ACE in 297 SARS-CoV-2-positive and 253 SARS-CoV-2-negative tested patients. We analyzed the association of the SNPs with susceptibility for SARS-CoV-2 infection and the severity of COVID-19. RESULTS: SARS-CoV-2-positive and SARS-CoV-2-negative patients did not differ regarding demographics and clinical characteristics. For ACE2 rs2285666, the GG genotype or G-allele was significantly associated with an almost two-fold increased SARS-CoV-2 infection risk and a three-fold increased risk to develop serious disease or COVID-19 fatality. In contrast, the ACE polymorphism was not related to infection risk or severity of disease. In a multivariable analysis, the ACE2 rs2285666 G-allele remained as an independent risk factor for serious disease besides the known risk factors male gender and cardiovascular disease. CONCLUSIONS: In summary, our report appears to be the first showing that a common ACE2 polymorphism impacts the risk for SARS-CoV-2 infection and the course of COVID-19 independently from previously described risk factors.

NAT2 polymorphisms as a cause of metamizole-induced agranulocytosis.

Metamizole is a widely prescribed NSAID with excellent analgesic and antipyretic properties. Although very effective, it is banned in some countries because of the risk for severe agranulocytosis. We here describe three patients with metamizole-associated agranulocytosis. Patient #1 suffered from agranulocytosis and tonsillitis followed by severe sepsis by Streptococcus pneumoniae and Epstein-Barr virus reactivation. Her dizygotic twin sister (patient #2) also suffered from agranulocytosis after a surgical intervention. Patient #3 initially had a tonsillitis and also developed neutropenia after metamizole intake. For all patients, pharmacogenetic diagnostic for the genes CYP2C9, CYP2C19 and NAT2, which are involved in metamizole metabolism and degradation of toxic metabolites, was initiated. Pharmacogenetic analysis revealed NAT2 slow acetylator phenotype in all three patients. Additionally, patient #2 is an intermediate metabolizer for CYP2C19 and patient #3 is a poor metabolizer for CYP2C9. Impairment of these enzymes causes a reduced degradation of toxic metabolites, for example, 4-methylaminoantipyrine (4-MAA) or 4-aminoantipyrine. The metabolite 4-MAA can complex with hemin, which is an early breakdown product during hemolysis. Hemolysis is often observed during invasive infections or after surgical procedures. It is known that the 4-MAA/hemin complex can induce cytotoxicity in the bone marrow and interrupt granulocyte maturation. In conclusion, metamizole-induced agranulocytosis most likely was a consequence of the underlying genetical predisposition, that is, polymorphisms in the genes NAT2, CYP2C9 and CYP2C19. Hemolysis may have increased the toxicity of metamizole metabolites.

The influence of IFITM3 polymorphisms on susceptibility to SARS-CoV-2 infection and severity of COVID-19.

BACKGROUND AND AIMS: The interferon-induced transmembrane protein 3 (IFITM3) plays an important role in the adaptive and innate immune response by inhibiting viral membrane hemifusion between the host and viral cell cytoplasm. Single nucleotide polymorphisms (SNPs) in the gene IFITM3 have been associated with susceptibility and severity of influenza or other viral infections. We aimed to analyze the role of SNPs in the gene IFITM3 in SARS-CoV-2 infection. METHODS: We performed genotyping of the SNPs rs12252 and rs34481144 in the gene IFITM3 in 239 SARS-CoV-2-positive and 253 SARS-CoV-2-negative patients. We analyzed the association of the SNPs with susceptibility to SARS-CoV-2 infection and severity of COVID-19. RESULTS: SARS-CoV-2-positive and SARS-CoV-2-negative patients did not differ regarding demographics. Neither IFITM3 rs12252 nor rs34481144 polymorphisms were related to SARS-CoV-2 infection risk or severity of COVID-19. Interestingly, we observed the putative deleterious rs12252 CC genotype only in SARS-CoV-2-positive patients (N = 2). Also, we found a non-significant higher frequency of rs34481144 A-allele carriers in the patients with 'serious' COVID-19. CONCLUSIONS: In summary, we could not confirm the recently reported influence of polymorphisms in the gene IFITM3 on SARS-CoV-2 infection risk or severity of COVID-19 in a German cohort. Additional studies are needed to clarify the influence of the rs12252 CC genotype on SARS-CoV-2 infection risk and the rs34481144 A-allele on course of COVID-19.

Characterization of the G protein-coupled receptor kinase 6 promoter reveals a functional CREB binding site.

BACKGROUND: G protein-coupled receptor kinase 6 (GRK6) is part of the G protein-coupled receptor kinase family, whose members act as key regulators of seven-transmembrane receptor signalling. GRK6 seems to play a role in regulation of inflammatory processes, but mechanisms of transcriptional regulation of GRK6 expression in inflammatory cell lines have not been characterized. Protein kinase C (PKC) signalling is also involved in inflammatory regulation and an impact of PKC activation on GRK6 protein expression was described previously. Thus, the aim of this study was to 1) characterize the GRK6 promoter, and 2) investigate a potential influence of PKC on GRK6 expression. METHODS: Five deletion constructs of the GRK6 promoter were cloned. After transient transfection into a human T cell line, promoter activity was assessed using luciferase reporter gene assays. Putative transcription factor binding sites were identified, mutated, and binding was investigated using electrophoretic mobility shift assays (EMSA). Following stimulation with a PKC activator, GRK6 expression on mRNA and protein levels was assessed by reverse transcriptase qPCR and Western blots. RESULTS: Investigation of the GRK6 promoter revealed a putative cAMP responsive element (CRE), whose mutation led to decreased promoter activity (p = 0.0006). Functionality of the CRE binding protein (CREB) binding site was verified in EMSA blots. Stimulation with a PKC activator resulted in decreased GRK6 promoter activity (p = 0.0027), mRNA (p = 0.04) and protein expression. CONCLUSION: We characterized the human GRK6 promoter and identified promoter activity to be influenced by a CREB binding site. PKC might be one determinant contributing to altered GRK6 expression.

Lack of association of common polymorphisms linked to empathic behavior with self-reported trait empathy in healthy volunteers.

BACKGROUND: In a previously specified sample of 421 healthy subjects, we found associations of a common oxytocin receptor (OXTR) polymorphism with self-reported trait empathy. In this study, we used this sample to explore polymorphisms in other genes which have been frequently linked to empathic behavior for associations with self-reported trait empathy: CD38 (CD38), involved in oxytocin secretion, the serotonin transporter (SLC6A4), the Catechol-O-Methyltransferase (COMT) and the corticotropin releasing hormone receptor 1 (CRHR1). METHODS: We genotyped our sample for the following common polymorphisms: rs3796863 in the CD38 gene, 5-HTTLPR in the SLC6A4 gene, rs4680 in the COMT gene and rs242924 in the CRHR1 gene. Dispositional empathy was tested using Davis' Interpersonal Reactivity Index (IRI). We used a Bonferroni corrected alpha level of p = 0.002 to adjust for multiple comparisons. RESULTS: None of the genotypes were associated with any of the IRI scales for the complete sample (n = 421) or for the sub-groups of male (n = 213) and female (n = 190) participants. Our sample of 421 participants achieved 95% power to detect effects greater than r = ±0.18. For smaller effects, however, false negatives could not be rejected with equal confidence as false positives. CONCLUSIONS: We conclude that an association between the four polymorphisms with trait empathy measured by the IRI may not be present. We propose that the associations that have been found in other studies can be largely explained by differences in empathy-related constructs and measurements.

Genetic variations in G-protein signal pathways influence progression of coronary artery calcification: Results from the Heinz Nixdorf Recall study.

BACKGROUND AND AIMS: Coronary artery calcification (CAC) is one of the most sensitive and specific markers of coronary atherosclerosis and believed to be heritable. We hypothesized that functionally relevant single-nucleotide polymorphisms (SNPs) in the G-protein signal pathway, which have been previously related to coronary artery disease, are associated with CAC progression. METHODS: 3108 participants from the Heinz Nixdorf Recall study with CAC measurements at both baseline (CACb) and 5-year follow-up (CAC5y) were included. We genotyped SNPs rs1042714 (ADRB2), rs6026584 and rs12481583 (GNAS), and rs5443 (GNB3) and defined a priori risk alleles derived from literature data. Regression analyses were applied to measures of 5-year CAC progression, unadjusted, adjusted for age, sex, and adjusted for age, sex, log(CACb+1) as well as for cardiovascular risk factors. RESULTS: The presence of one or more risk alleles was associated with a 26.9% (95% CI 5.5-52.4) increase in 5-year CAC progression (p = 0.011) and a 29.2% (95% CI 5.9-57.6) accelerated increase of CAC over the 5-year period compared to what was expected with respect to the baseline CAC percentile value (p = 0.012). Each of those risk alleles increased the 5-year CAC progression by 4.4% (95% CI 1.3-7.6, p = 0.006) and resulted in a 4.9% accelerated increase of CAC over the 5-year period (95% CI 1.6-8.4, p = 0.004). These unadjusted data did not change after adjustment. CONCLUSIONS: Genetic variations in the G-protein signal pathway are associated with CAC progression in a cumulative fashion, indicating the importance of the pathway for genetic heritability in CAC progression and coronary artery disease.

Major Adverse Kidney Events Are Associated with the Aquaporin 5 -1364A/C Promoter Polymorphism in Sepsis: A Prospective Validation Study.

Since the functionally important AQP5 -1364A/C single nucleotide promoter polymorphism alters key mechanisms of inflammation and survival in sepsis, it may affect the risk of an acute kidney injury. Accordingly, we tested the hypothesis in septic patients that this AQP5 polymorphism is associated with major adverse kidney events and also validated its impact on 90-day survival. In this prospective observational monocentric genetic association study 282 septic patients were included and genotyped for the AQP5 -1364A/C polymorphism (rs3759129). The primary endpoint was the development of major adverse kidney events within 30 days. In AC/CC genotypes, major adverse kidney events were less frequent (41.7%) than in AA genotypes (74.3%; OR:0.34; 95%-CI: 0.18-0.62; p < 0.001). Ninety-day survival was also associated with the AQP5 polymorphism (p = 0.004), with 94/167 deaths (56.3%) in AA genotypes, but only 46/115 deaths (40.0%) in C-allele carriers. Multiple proportional hazard analysis revealed AC/CC genotypes to be at significantly lower risk for death within 90 days (HR: 0.60; 95%-CI: 0.42-0.86; p = 0.006). These findings confirm the important role of the AQP5 -1364A/C polymorphism as an independent prognostic factor in sepsis. Furthermore, we demonstrate a strong association between this AQP5 polymorphism and susceptibility for major adverse kidney events suggesting a promising characteristic in terms of precision medicine.

DNA methylation of a NF-κB binding site in the aquaporin 5 promoter impacts on mortality in sepsis.

Altered aquaporin 5 (AQP5) expression in immune cells impacts on key mechanisms of inflammation and is associated with sepsis survival. Since epigenetic regulation via DNA methylation might contribute to a differential AQP5 expression in sepsis, we tested the hypotheses that DNA methylation of the AQP5 promotor (1) influences AQP5 expression, (2) is associated with the 30-day survival of septic patients, and (3) alters the nuclear transcription factor NF-κB binding. AQP5 mRNA expression was quantified by real-time PCR in whole blood samples of 135 septic patients. In silico computer analysis of the AQP5 promoter (nt-567 to nt-975) revealed seven putative inflammatory transcription factor binding sites and methylation of these sites was analyzed. Electrophoretic mobility shift assays were performed to assess the binding of nuclear NF-κB to the AQP5 promoter region nt-937. After adjustment for multiple testing, a greater methylation rate was found at cytosine site nt-937 in the AQP5 promoter linked to NF-κB binding in non-survivors compared to survivors (p = 0.002, padj = 0.014). This was associated with greater AQP5 mRNA expression in non-survivors (p = 0.037). Greater (≥16%) promoter methylation at nt-937 was also associated with an independently increased risk of death within 30 days (HR: 3.31; 95% CI: 1.54-6.23; p = 0.002). We detected a functionally important AQP5 promoter cytosine site (nt-937) linked to the binding of the inflammatorily acting nuclear transcription factor NF-κB, with increased methylation in sepsis non-survivors. Thus, nt-937 APQ5 promoter methylation, presumably related to NF-κB binding, is prognostically relevant in sepsis and demonstrates that epigenetic changes impact on sepsis outcome.

The GNAS SNP c.393C>T (rs7121) as a marker for disease progression and survival in cancer.

G-protein receptor signaling plays a key role in multiple signal transduction pathways. Aberrant activity of the stimulatory Gsα subunit has been frequently associated with cancer. GNAS sequence alterations and conformational changes of Gsα can both enhance or diminish its function and change downstream effects of G-protein receptor signaling. In this review and meta-analysis, we focus on the synonymous SNP rs7121 (FokI, c.393C>T), which is associated with either tumor progression or prolonged survival in cancer patients (overall hazard ratio = 2.256; p < 0.001). We finally point out the relevance of GNAS rs7121 as a promising biomarker and a prediction tool for therapy response and the need of further experiments to implement it into routine clinical diagnostics.