Hanneke W. M. van Deutekom

A mutation in the glutamate-rich region of RNA-binding motif protein 20 causes dilated cardiomyopathy through missplicing of titin and impaired Frank–Starling mechanism

AIM Mutations in the RS-domain of RNA-binding motif protein 20 (RBM20) have recently been identified to segregate with aggressive forms of familial dilated cardiomyopathy (DCM). Loss of RBM20 in rats results in missplicing of the sarcomeric gene titin (TTN). The functional and physiological consequences of RBM20 mutations outside the mutational hotspot of RBM20 have not been explored to date. In this study, we investigated the pathomechanism of DCM caused by a novel RBM20 mutation in human cardiomyocytes. METHODS AND RESULTS We identified a family with DCM carrying a mutation (RBM20(E913K/+)) in a glutamate-rich region of RBM20. Western blot analysis of endogenous RBM20 protein revealed strongly reduced protein levels in the heart of an RBM20(E913K/+ )carrier. RNA deep-sequencing demonstrated massive inclusion of exons coding for the spring region of titin in the RBM20(E913K/+ )carrier. Titin isoform analysis revealed a dramatic shift from the less compliant N2B towards the highly compliant N2BA isoforms in RBM20(E913K/+ )heart. Moreover, an increased sarcomere resting-length was observed in single cardiomyocytes and isometric force measurements revealed an attenuated Frank-Starling mechanism (FSM), which was rescued by protein kinase A treatment. CONCLUSION A mutation outside the mutational hotspot of RBM20 results in haploinsufficiency of RBM20. This leads to disturbed alternative splicing of TTN, resulting in a dramatic shift to highly compliant titin isoforms and an impaired FSM. These effects may contribute to the early onset, and malignant course of DCM caused by RBM20 mutations. Altogether, our results demonstrate that heterozygous loss of RBM20 suffices to profoundly impair myocyte biomechanics by its disturbance of TTN splicing.

The Rate of Immune Escape Vanishes When Multiple Immune Responses Control an HIV Infection

During the first months of HIV infection, the virus typically evolves several immune escape mutations. These mutations are found in epitopes in viral proteins and reduce the impact of the CD8+ T cells specific for these epitopes. Recent data show that only a subset of the epitopes escapes, that most of these escapes evolve early, and that the rate of immune escape slows down considerably. To investigate why the evolution of immune escape slows down over the time of infection, we have extended a consensus mathematical model to allow several immune responses to control the virus together. In the extended model, most escapes also occur early, and the immune escape rate becomes small later, and typically only a minority of the epitopes escape. We show that escaping one of the many immune responses provides little advantage after viral setpoint has been approached because the total killing rate hardly depends on the breadth of the immune response. If the breadth of the immune response slowly wanes during disease progression, the model predicts an increase in the rate of immune escape at late stages of infection. Overall, the most striking prediction of the model is that HIV evolves a small number of immune escapes, in both relative and absolute terms, when the CTL immune response is broad.

Loss of Hiv-1-derived cytotoxic T lymphocyte epitopes restricted by protective Hla-b alleles during the Hiv-1 epidemic

Objective and design:HIV-1 is known to adapt to the human immune system, leading to accumulation of escape mutations during the course of infection within an individual. Cross-sectional studies have shown an inverse correlation between the prevalence of human leukocyte antigen (HLA) alleles in a population and the number of cytotoxic T lymphocyte (CTL) escape mutations in epitopes restricted by those HLA alleles. Recently, it was demonstrated that at a population level HIV-1 is adapting to the humoral immune response, which is reflected in an increase in resistance to neutralizing antibodies over time. Here we investigated whether adaptations to cellular immunity have also accumulated during the epidemic. Methods:We compared the number of CTL epitopes in HIV-1 strains isolated from individuals who seroconverted at the beginning of the HIV-1 epidemic and from individuals who seroconverted in recent calendar time. Results:The number of CTL epitopes in HIV-1 variants restricted by the most common HLA alleles in the population did not change significantly during the epidemic. In contrast, we found a significant loss of CTL epitopes restricted by HLA-B alleles associated with a low relative hazard of HIV-1 disease progression during the epidemic. Such a loss was not observed for CTL epitopes restricted by HLA-A alleles. Conclusion:Despite the large degree of HLA polymorphism, HIV-1 has accumulated adaptations to CTL responses within 20 years of the epidemic. The fact that such adaptations are driven by the HLA-B molecules that provide best protection against HIV-1 disease progression has important implications for our understanding of HIV evolution.

Refinement of the definition of permissible HLA-DPB1 mismatches with predicted indirectly recognizable HLA-DPB1 epitopes.

Hematopoietic stem cell transplantation with HLA-DPB1-mismatched donors leads to an increased risk of acute graft-versus-host disease (GVHD). Studies have indicated a prognostic value for classifying HLA-DPB1 mismatches based on T cell-epitope (TCE) groups. The aim of this study was to determine the contribution of indirect recognition of HLA-DP-derived epitopes, as determined with the Predicted Indirectly ReCognizable HLA Epitopes (PIRCHE) method. We therefore conducted a retrospective single-center analysis on 80 patients transplanted with a 10/10 matched unrelated donor that was HLA-DPB1 mismatched. HLA-DPB1 mismatches that were classified as GVH nonpermissive by the TCE algorithm correlated to higher numbers of HLA class I as well as HLA class II presented PIRCHE (PIRCHE-I and -II) compared with permissive or host-versus-graft nonpermissive mismatches. Patients with acute GVHD grades II to IV presented significantly higher numbers of PIRCHE-I compared with patients without acute GVHD (P < .05). Patients were divided into 2 groups based on the presence or absence of PIRCHE. Patients with PIRCHE-I or -II have an increased hazard of acute GVHD when compared with patients without PIRCHE-I or -II (hazard ratio [HR], 3.19; 95% confidence interval [CI], 1.10 to 9.19; P < .05; and HR, 4.07; 95% CI, .97 to 17.19; P = .06, respectively). Patients classified as having an HLA-DPB1 permissive mismatch by the TCE model had an increased risk of acute GVHD when comparing presence of PIRCHE-I with absence of PIRCHE-I (HR, 2.96; 95% CI, .84 to 10.39; P = .09). We therefore conclude that the data presented in this study describe an attractive and feasible possibility to better select permissible HLA-DPB1 mismatches by including both a direct and an indirect recognition model.

Atrial Fibrillation Is an Independent Risk Factor for Ventricular Fibrillation A Large-Scale Population-Based Case-Control Study

Background—Atrial fibrillation (AF) is associated with sudden cardiac death. We aimed to study whether AF is associated with ventricular fibrillation (VF), the most common cause of sudden cardiac death and whether this association is independent of confounders, ie, concomitant disease, use of antiarrhythmic or QT-prolonging drugs, and acute myocardial infarction. Methods and Results—We performed a community-based case-control study. Cases were patients with out-of-hospital cardiac arrest because of ECG-documented VF. Controls were age-/sex-matched non-VF subjects from the community. VF risk in AF patients was studied by means of (conditional) logistic regression, adjusting for all available confounders. We studied 1397 VF cases and 3474 controls. AF occurred in 215 cases (15.4%) and 90 controls (2.6%). AF was associated with a 3-fold increased risk of VF (adjusted odds ratio, 3.1 [2.1–4.5]). VF risk in AF cases was increased to the same extent across all age/sex groups and in AF cases who had no comorbidity (adjusted odds ratio 3.0 [1.6–5.5]) or used no confounding drugs (antiarrhythmics, 2.4 [1.4–4.3]; QT-prolonging drugs, 3.1 [1.8–5.4]). VF risk was similarly increased in AF cases with acute myocardial infarction–related VF (adjusted odds ratio 2.6 [1.4–4.8]), and those with non-acute myocardial infarction–related VF (adjusted odds ratio 4.3 [1.9–10.1]). Conclusions—AF is independently associated with a 3-fold increased risk of VF. Comorbidity, use of antiarrhythmic or QT-prolonging drugs, or acute myocardial infarction does not fully account for this increased risk.Background— Atrial fibrillation (AF) is associated with sudden cardiac death. We aimed to study whether AF is associated with ventricular fibrillation (VF), the most common cause of sudden cardiac death and whether this association is independent of confounders, ie, concomitant disease, use of antiarrhythmic or QT-prolonging drugs, and acute myocardial infarction. Methods and Results— We performed a community-based case-control study. Cases were patients with out-of-hospital cardiac arrest because of ECG-documented VF. Controls were age-/sex-matched non-VF subjects from the community. VF risk in AF patients was studied by means of (conditional) logistic regression, adjusting for all available confounders. We studied 1397 VF cases and 3474 controls. AF occurred in 215 cases (15.4%) and 90 controls (2.6%). AF was associated with a 3-fold increased risk of VF (adjusted odds ratio, 3.1 [2.1–4.5]). VF risk in AF cases was increased to the same extent across all age/sex groups and in AF cases who had no comorbidity (adjusted odds ratio 3.0 [1.6–5.5]) or used no confounding drugs (antiarrhythmics, 2.4 [1.4–4.3]; QT-prolonging drugs, 3.1 [1.8–5.4]). VF risk was similarly increased in AF cases with acute myocardial infarction–related VF (adjusted odds ratio 2.6 [1.4–4.8]), and those with non-acute myocardial infarction–related VF (adjusted odds ratio 4.3 [1.9–10.1]). Conclusions— AF is independently associated with a 3-fold increased risk of VF. Comorbidity, use of antiarrhythmic or QT-prolonging drugs, or acute myocardial infarction does not fully account for this increased risk.

Zooming into the binding groove of HLA molecules: which positions and which substitutions change peptide binding most?

Human leukocyte antigen (HLA) genes are the most polymorphic genes in the human genome. Almost all polymorphic residues are located in the peptide-binding groove, resulting in different peptide-binding preferences. Whether a single amino acid change can alter the peptide-binding repertoire of an HLA molecule has never been shown. To experimentally quantify the contribution of a single amino acid change to the peptide repertoire of even a single HLA molecule requires an immense number of HLA peptide-binding measurements. Therefore, we used an in silico method to study the effect of single mutations on the peptide repertoires. We predicted the peptide-binding repertoire of a large set of HLA molecules and used the overlap of the peptide-binding repertoires of each pair of HLA molecules that differ on a single position to measure how much single substitutions change the peptide binding. We found that the effect of a single substitution in the peptide-binding groove depends on the substituted position and the amino acids involved. The positions that alter peptide binding most are the most polymorphic ones, while those that are hardly variable among HLA molecules have the lowest effect on the peptide repertoire. Although expected, the relationship between functional divergence and polymorphism of HLA molecules has never been shown before. Additionally, we show that a single substitution in HLA-B molecules has more effect on the peptide-binding repertoire compared to that in HLA-A molecules. This provides an (alternative) explanation for the larger polymorphism of HLA-B molecules compared to HLA-A molecules.

A Comparative Analysis of Viral Peptides Presented by Contemporary Human and Chimpanzee MHC Class I Molecules

Genetic factors such as the MHC influence the immunocompetence of an individual. MHC genes are the most polymorphic genes in primates, which is often interpreted as an adaptation to establish good T cell responses to a wide range of (evolving) pathogens. Chimpanzee MHC (Patr) genes are less polymorphic than human MHC (HLA) genes, which is surprising because chimpanzee is the older species of the two and is therefore expected to display more variation. To quantify the effect of the reduced polymorphism, we compared the peptide binding repertoire of human and chimpanzee MHC molecules. Using a peptide-MHC binding predictor and proteomes of >900 mammalian viruses, we show that, at the population level, the total peptide binding repertoire of Patr-A molecules is ∼36% lower than that of their human counterparts, whereas the reduction of the peptide binding repertoire of the Patr-B locus is only 15%. In line with these results, different Patr-A molecules turn out to have largely overlapping peptide binding repertoires, whereas the Patr-B molecules are more distinct from each other. This difference is somewhat less apparent at the individual level, where we found that only 25% of the viruses are significantly better presented by “simulated” humans with heterozygous HLA-A and -B loci. Taken together, our results indicate that the Patr-B molecules recovered more after the selective sweep, whereas the Patr-A locus shows the most signs of the selective sweep with regard to its peptide binding repertoire.

Natural genetic variation of the cardiac transcriptome in non-diseased donors and patients with dilated cardiomyopathy

BackgroundGenetic variation is an important determinant of RNA transcription and splicing, which in turn contributes to variation in human traits, including cardiovascular diseases.ResultsHere we report the first in-depth survey of heart transcriptome variation using RNA-sequencing in 97 patients with dilated cardiomyopathy and 108 non-diseased controls. We reveal extensive differences of gene expression and splicing between dilated cardiomyopathy patients and controls, affecting known as well as novel dilated cardiomyopathy genes. Moreover, we show a widespread effect of genetic variation on the regulation of transcription, isoform usage, and allele-specific expression. Systematic annotation of genome-wide association SNPs identifies 60 functional candidate genes for heart phenotypes, representing 20% of all published heart genome-wide association loci. Focusing on the dilated cardiomyopathy phenotype we found that eQTL variants are also enriched for dilated cardiomyopathy genome-wide association signals in two independent cohorts.ConclusionsRNA transcription, splicing, and allele-specific expression are each important determinants of the dilated cardiomyopathy phenotype and are controlled by genetic factors. Our results represent a powerful resource for the field of cardiovascular genetics.

HIV-1 evolution in patients undergoing immunotherapy with Tat, Rev, and Nef expressing dendritic cells followed by treatment interruption

Objectives:This study aimed to evaluate HIV sequence evolution in whole genes and in CD8+ T-cell epitope regions following immunotherapy and subsequent analytical treatment interruption (ATI). A second objective of this study was to analyze associations between vaccine-specific immune responses and epitope mutation rates. Design:HIV-1-infected patients on combined antiretroviral therapy (cART) were subjected to immunotherapy by the administration of an autologous dendritic cell-based therapeutic vaccine expressing Tat, Rev, and Nef and subsequent ATI. Methods:HIV-1 genes were amplified and sequenced from plasma RNA obtained before initiation of cART as well as during ATI. Control sequences for virus evolution in untreated HIV-1-infected individuals were obtained from the HIV Sequence Database (Los Alamos). CD8+ T-cell epitope regions were defined based on literature data and prediction models. HIV-1-specific immune responses were evaluated to analyze their impact on sequence evolution. Results:Viral sequence evolution in the tat, rev, and nef genes of vaccinated patients was similar to that of controls. The number of mutations observed inside and outside CD8+ T-cell epitopes was comparable for vaccine-targeted and nontargeted proteins. We found no evidence for an impact of vaccine-induced or enhanced immune responses on the number of mutations inside or outside epitopes. Conclusion:Therapeutic vaccination of HIV-1-infected patients with a dendritic cell-based vaccine targeting Tat, Rev, and Nef did not affect virus evolution at the whole gene level nor at the CD8+ T-cell epitope level.

Atrial Fibrillation Is an Independent Risk Factor for Ventricular FibrillationCLINICAL PERSPECTIVE

Background—Atrial fibrillation (AF) is associated with sudden cardiac death. We aimed to study whether AF is associated with ventricular fibrillation (VF), the most common cause of sudden cardiac death and whether this association is independent of confounders, ie, concomitant disease, use of antiarrhythmic or QT-prolonging drugs, and acute myocardial infarction. Methods and Results—We performed a community-based case-control study. Cases were patients with out-of-hospital cardiac arrest because of ECG-documented VF. Controls were age-/sex-matched non-VF subjects from the community. VF risk in AF patients was studied by means of (conditional) logistic regression, adjusting for all available confounders. We studied 1397 VF cases and 3474 controls. AF occurred in 215 cases (15.4%) and 90 controls (2.6%). AF was associated with a 3-fold increased risk of VF (adjusted odds ratio, 3.1 [2.1–4.5]). VF risk in AF cases was increased to the same extent across all age/sex groups and in AF cases who had no comorbidity (adjusted odds ratio 3.0 [1.6–5.5]) or used no confounding drugs (antiarrhythmics, 2.4 [1.4–4.3]; QT-prolonging drugs, 3.1 [1.8–5.4]). VF risk was similarly increased in AF cases with acute myocardial infarction–related VF (adjusted odds ratio 2.6 [1.4–4.8]), and those with non-acute myocardial infarction–related VF (adjusted odds ratio 4.3 [1.9–10.1]). Conclusions—AF is independently associated with a 3-fold increased risk of VF. Comorbidity, use of antiarrhythmic or QT-prolonging drugs, or acute myocardial infarction does not fully account for this increased risk.Background— Atrial fibrillation (AF) is associated with sudden cardiac death. We aimed to study whether AF is associated with ventricular fibrillation (VF), the most common cause of sudden cardiac death and whether this association is independent of confounders, ie, concomitant disease, use of antiarrhythmic or QT-prolonging drugs, and acute myocardial infarction. Methods and Results— We performed a community-based case-control study. Cases were patients with out-of-hospital cardiac arrest because of ECG-documented VF. Controls were age-/sex-matched non-VF subjects from the community. VF risk in AF patients was studied by means of (conditional) logistic regression, adjusting for all available confounders. We studied 1397 VF cases and 3474 controls. AF occurred in 215 cases (15.4%) and 90 controls (2.6%). AF was associated with a 3-fold increased risk of VF (adjusted odds ratio, 3.1 [2.1–4.5]). VF risk in AF cases was increased to the same extent across all age/sex groups and in AF cases who had no comorbidity (adjusted odds ratio 3.0 [1.6–5.5]) or used no confounding drugs (antiarrhythmics, 2.4 [1.4–4.3]; QT-prolonging drugs, 3.1 [1.8–5.4]). VF risk was similarly increased in AF cases with acute myocardial infarction–related VF (adjusted odds ratio 2.6 [1.4–4.8]), and those with non-acute myocardial infarction–related VF (adjusted odds ratio 4.3 [1.9–10.1]). Conclusions— AF is independently associated with a 3-fold increased risk of VF. Comorbidity, use of antiarrhythmic or QT-prolonging drugs, or acute myocardial infarction does not fully account for this increased risk.