Search

Purpose: The genetic architecture of Plakophilin 2 (PKP2) cardiomyopathy can inform our understanding of its variant pathogenicity and protein function., Methods: We assess the gene-wide and regional association of truncating and missense variants in PKP2 with arrhythmogenic cardiomyopathy (ACM), and arrhythmogenic right ventricular cardiomyopathy (ARVC) specifically. A discovery data set compares genetic testing requisitions to gnomAD. Validation is performed in a rigorously phenotyped definite ARVC cohort and non-ACM individuals in the Geisinger MyCode cohort., Results: The etiologic fraction (EF) of ACM-related diagnoses from truncating variants in PKP2 is significant (0.85 [0.80,0.88], p < 2 × 10 -16 ), increases for ARVC specifically (EF = 0.96 [0.94,0.97], p < 2 × 10 -16 ), and is highest in definite ARVC versus non-ACM individuals (EF = 1.00 [1.00,1.00], p < 2 × 10 -16 ). Regions of missense variation enriched for ACM probands include known functional domains and the C-terminus, which was not previously known to contain a functional domain. No regional enrichment was identified for truncating variants., Conclusion: This multicohort evaluation of the genetic architecture of PKP2 demonstrates the specificity of PKP2 truncating variants for ARVC within the ACM disease spectrum. We identify the PKP2 C-terminus as a potential functional domain and find that truncating variants likely cause disease irrespective of transcript position., (© 2021. The Author(s).)

In the 8 years since the discovery of the genetic bases of catecholaminergic polymorphic ventricular tachycardia (CPVT), we have witnessed a remarkable improvement of knowledge on arrhythmogenic mechanisms involving disruption of cardiac Ca(2+) homeostasis. Studies on the consequences of RyR2 and CASQ2 mutations in cellular systems and mouse models have shed new light on pathways that are also implicated in arrhythmias occurring in highly prevalent diseases, such as heart failure. This research track has also led to the identification of therapeutic targets of potential clinical impact to abate the burden of sudden death in CPVT. Here, we review the current knowledge on the pathophysiology of CPVT also highlighting the existing controversies and possible future development.

Mice infected with the protozoan parasite, Trypanosoma cruzi, are known to be immunosuppressed in responsiveness to heterologous antigens and parasite-specific antigens. This suppression is mediated by suppressor macrophages and is exemplified by deficient T cell activity and abnormal cytokine production. Neither the mechanism by which suppressor macrophages effect suppression nor the characteristics of these suppressor macrophages is known. In the present study, we analyzed the regulatory cell populations in splenocytes of infected mice (SCinf) and their interactions by limiting dilution-partition analysis, an approach which allows the functional separation of multiple regulatory cell subpopulations within cell mixtures. Our results demonstrate the presence of a complex immunoregulatory circuit in SCinf affecting the generation of anti-sheep erythrocyte antibody responses in vitro. Titration of SCinf (but not peritoneal exudate or lymph node cells) into Mishell-Dutton microcultures of normal spleen cells generated complex dose-response curves with two zones of suppressed responses following the addition of either low or high doses of SCinf to the cultures. Addition of intermediate doses of SCinf to the microcultures restored responsiveness. Both the low- and high-dose zones of suppression were shown to be mediated by macrophages, whereas T cells were responsible for the restored responsiveness at intermediate doses of SCinf. Examination of the development of this complex regulatory pattern during the course of the acute phase of infection indicated the sequential development of one suppressor macrophage population, followed by the development of the beneficial T cell population, and finally the expression of the second suppressive macrophage population.

Limiting dilution analysis has been a valuable approach for both determining the frequency of cell subpopulations elicited during immune responses, as well as for the analysis of immunoregulatory circuits. We describe a simple, visually scored spot test for evaluating the response of Mishell-Dutton microcultures used in limiting dilution analysis. This spot test is based on a microELISA using immunoreagents conjugated to the enzyme, urease, as an alternative to the hemolytic spot test. The assay as performed in Terasaki trays requires minute quantities (less than 10 microliters) of culture supernatant, yet the ELISA yields a distinct color difference between tray wells containing culture supernatants derived from responding (purple) and nonresponding (yellow) microcultures. Although designed to be scored rapidly by visual inspection, the assay can be quantified by manual alignment of the Terasaki tray wells on commercially available ELISA plate readers with an accuracy and reproducibility comparable to assays performed in 96-well ELISA plates. Determination of anti-sheep RBC responses in limiting dilution Mishell-Dutton microcultures with both the hemolytic spot test and the urease-microELISA spot test showed a very close correlation between the results of the two assays. However, the urease-microELISA should be amenable for use with antigens not readily conjugated to an indicator RBC, and should be useful in those situations where determination of the antibody subclass(es) produced by responding microcultures is desired.