Mapping the dynamics of epigenetic adaptation during heterochromatin misregulation.

A classical and well-established mechanism that enables cells to adapt to new and adverse conditions is the acquisition of beneficial genetic mutations. Much less is known about epigenetic mechanisms that allow cells to develop novel and adaptive phenotypes without altering their genetic blueprint. It has been recently proposed that histone modifications, such as heterochromatin-defining H3K9 methylation (H3K9me), normally reserved to maintain genome integrity, can be redistributed across the genome to establish new and potentially adaptive phenotypes. To uncover the dynamics of this process, we developed a precision engineered genetic approach to trigger H3K9me redistribution on-demand in fission yeast. This enabled us to trace genome-scale RNA and chromatin changes over time prior to and during adaptation in long-term continuous cultures. Establishing adaptive H3K9me occurs over remarkably slow time-scales relative to the initiating stress. During this time, we captured dynamic H3K9me redistribution events ultimately leading to cells converging on an optimal adaptive solution. Upon removal of stress, cells relax to new transcriptional and chromatin states rather than revert to their initial (ground) state, establishing a tunable memory for a future adaptive epigenetic response. Collectively, our tools uncover the slow kinetics of epigenetic adaptation that allow cells to search for and heritably encode adaptive solutions, with implications for drug resistance and response to infection.

Genetic Contribution to End-Stage Cardiomyopathy Requiring Heart Transplantation.

BACKGROUND: Many cardiovascular disorders propel the development of advanced heart failure that necessitates cardiac transplantation. When treatable causes are excluded, studies to define causes are often abandoned, resulting in a diagnosis of end-stage idiopathic cardiomyopathy. We studied whether DNA sequence analyses could identify unrecognized causes of end-stage nonischemic cardiomyopathy requiring heart transplantation and whether the prevalence of genetic causes differed from ambulatory cardiomyopathy cases. METHODS: We performed whole exome and genome sequencing of 122 explanted hearts from 101 adult and 21 pediatric patients with idiopathic cardiomyopathy from a single center. Data were analyzed for pathogenic/likely pathogenic variants in nuclear and mitochondrial genomes and assessed for nonhuman microbial sequences. The frequency of damaging genetic variants was compared among cardiomyopathy cohorts with different clinical severity. RESULTS: Fifty-four samples (44.3%) had pathogenic/likely pathogenic cardiomyopathy gene variants. The frequency of pathogenic variants was similar in pediatric (42.9%) and adult (43.6%) samples, but the distribution of mutated genes differed (P=8.30×10-4). The prevalence of causal genetic variants was significantly higher in end-stage than in previously reported ambulatory adult dilated cardiomyopathy cases (P<0.001). Among remaining samples with unexplained causes, no damaging mitochondrial variants were identified, but 28 samples contained parvovirus genome sequences, including 2 samples with 6- to 9-fold higher levels than the overall mean levels in other samples. CONCLUSIONS: Pathogenic variants and viral myocarditis were identified in 45.9% of patients with unexplained end-stage cardiomyopathy. Damaging gene variants are significantly more frequent among transplant compared with patients with ambulatory cardiomyopathy. Genetic analyses can help define cause of end-stage cardiomyopathy to guide management and risk stratification of patients and family members.

Efficient in vivo genome editing prevents hypertrophic cardiomyopathy in mice.

Dominant missense pathogenic variants in cardiac myosin heavy chain cause hypertrophic cardiomyopathy (HCM), a currently incurable disorder that increases risk for stroke, heart failure and sudden cardiac death. In this study, we assessed two different genetic therapies-an adenine base editor (ABE8e) and a potent Cas9 nuclease delivered by AAV9-to prevent disease in mice carrying the heterozygous HCM pathogenic variant myosin R403Q. One dose of dual-AAV9 vectors, each carrying one half of RNA-guided ABE8e, corrected the pathogenic variant in ≥70% of ventricular cardiomyocytes and maintained durable, normal cardiac structure and function. An additional dose provided more editing in the atria but also increased bystander editing. AAV9 delivery of RNA-guided Cas9 nuclease effectively inactivated the pathogenic allele, albeit with dose-dependent toxicities, necessitating a narrow therapeutic window to maintain health. These preclinical studies demonstrate considerable potential for single-dose genetic therapies to correct or silence pathogenic variants and prevent the development of HCM.

Damaging variants in FOXI3 cause microtia and craniofacial microsomia.

PURPOSE: Craniofacial microsomia (CFM) represents a spectrum of craniofacial malformations, ranging from isolated microtia with or without aural atresia to underdevelopment of the mandible, maxilla, orbit, facial soft tissue, and/or facial nerve. The genetic causes of CFM remain largely unknown. METHODS: We performed genome sequencing and linkage analysis in patients and families with microtia and CFM of unknown genetic etiology. The functional consequences of damaging missense variants were evaluated through expression of wild-type and mutant proteins in vitro. RESULTS: We studied a 5-generation kindred with microtia, identifying a missense variant in FOXI3 (p.Arg236Trp) as the cause of disease (logarithm of the odds = 3.33). We subsequently identified 6 individuals from 3 additional kindreds with microtia-CFM spectrum phenotypes harboring damaging variants in FOXI3, a regulator of ectodermal and neural crest development. Missense variants in the nuclear localization sequence were identified in cases with isolated microtia with aural atresia and found to affect subcellular localization of FOXI3. Loss of function variants were found in patients with microtia and mandibular hypoplasia (CFM), suggesting dosage sensitivity of FOXI3. CONCLUSION: Damaging variants in FOXI3 are the second most frequent genetic cause of CFM, causing 1% of all cases, including 13% of familial cases in our cohort.

What can be done to improve diagnosis of aortic dissection?

Aortic dissection (AD) is rare. Missed AD is a common reason for coronial investigations and civil claims for medical negligence. Recommendations include improved education, supervision and information transfer, reminders in chest pain pathways and higher rates of investigation for AD. Higher investigation rates pose risks to patients and the health system which may not be in balance with the likelihood of AD. The appropriate diagnostic yield of investigation to balance risk and benefit has not been defined. The AD detection risk score pathway has been proposed as a useful diagnostic tool but concerns about its derivation, validation and utility remain. In this paper, we try to draw together published literature and local audit data to develop recommendations about what might be done to reduce the number of missed AD cases in EDs and what the impact of higher investigation rates might be.

An ancient founder mutation located between ROBO1 and ROBO2 is responsible for increased microtia risk in Amerindigenous populations.

Microtia is a congenital malformation that encompasses mild hypoplasia to complete loss of the external ear, or pinna. Although the contribution of genetic variation and environmental factors to microtia remains elusive, Amerindigenous populations have the highest reported incidence. Here, using both transmission disequilibrium tests and association studies in microtia trios (parents and affected child) and microtia cohorts enrolled in Latin America, we map an ∼10-kb microtia locus (odds ratio = 4.7; P = 6.78e-18) to the intergenic region between Roundabout 1 (ROBO1) and Roundabout 2 (ROBO2) (chr3: 78546526 to 78555137). While alleles at the microtia locus significantly increase the risk of microtia, their penetrance is low (<1%). We demonstrate that the microtia locus contains a polymorphic complex repeat element that is expanded in affected individuals. The locus is located near a chromatin loop region that regulates ROBO1 and ROBO2 expression in induced pluripotent stem cell­derived neural crest cells. Furthermore, we use single nuclear RNA sequencing to demonstrate ROBO1 and ROBO2 expression in both fibroblasts and chondrocytes of the mature human pinna. Because the microtia allele is enriched in Amerindigenous populations and is shared by some East Asian subjects with craniofacial malformations, we propose that both populations share a mutation that arose in a common ancestor prior to the ancient migration of Eurasian populations into the Americas and that the high incidence of microtia among Amerindigenous populations reflects the population bottleneck that occurred during the migration out of Eurasia.

Isolation of Nuclei from Mammalian Cells and Tissues for Single-Nucleus Molecular Profiling.

Both single-cell RNA sequencing (scRNAseq) and single-nucleus RNA sequencing (snRNAseq) can be used to characterize the transcriptional profile of individual cells, and based on these transcriptional profiles, help define cell type distribution in mixed cell populations. However, scRNAseq analyses are confounded if some of the cells are large (>50 µm) or if some of cells adhere more tightly to some adjacent cells than to others. Further, single cell isolation for scRNAseq requires fresh tissue, which may not be available for human or animal model tissues. Additionally, the current enzymatic and mechanical methods for single-cell dissociation can lead to stress-induced transcriptional artifacts. Nuclei for snRNAseq, on the other hand, can be isolated from any cell, regardless of size, and from either fresh or frozen tissues, and compared to whole cells, they are more resistant to mechanical pressures and appear not to exhibit as many cell isolation-based transcriptional artifacts. Here, we describe a time- and cost-effective procedure to isolate nuclei from mammalian cells and tissues. The protocol incorporates steps to mechanically disrupt samples to release nuclei. Compared to conventional nuclei isolation protocols, the approach described here increases overall efficiency, eliminates risk of contaminant exposure, and reduces volumes of expensive reagents. A series of RNA quality control checks are also incorporated to ensure success and reduce costs of subsequent snRNAseq experiments. Nuclei isolated by this procedure can be separated on the 10× Genomics Chromium system for either snRNAseq and/or Single-Nucleus ATAC-Seq (snATAC-Seq), and is also compatible with other single cell platforms. © 2021 Wiley Periodicals LLC. Basic Protocol 1: Sample preparation and quality control check via RNA Isolation and Analysis Basic Protocol 2: Nuclei Isolation.

National Coroners Information System: a valuable source of lessons for emergency medicine.

OBJECTIVE: To interrogate the National Coroners Information System (NCIS) to determine the recurrent themes among coroners' recommendations that aimed to increase the safety of ED care. METHODS: This was a retrospective analysis of NCIS closed cases, from Queensland, New South Wales, Tasmania, Victoria, Australian Capital Territory, South Australia and North Territory, entered since its inception in 2000. The keyword 'emergency department' returned 1645 cases, of which 180 were found to be relevant. The primary outcomes were the number and nature of cases where recommendations for improvements in ED care had been made and the recurrent themes of these recommendations that could inform education initiatives. RESULTS: Of the 180 cases, 108 (60.0%) were of deceased men and subject age ranged from 2 days to 91 years. The commonest causes of death were trauma (26.7%), infection (24.4%), cardiac events (15.0%) and poisoning (8.9%). No coronial recommendations were required in 19 cases. For the remainder, recommendation themes related to issues of risk management/medico-legal, diagnostic/therapeutic error, education, documentation/communication and re-presentation. The themes associated with the different doctor designations (consultant, registrar, resident/intern) were similar, although registrars and residents/interns tended towards more diagnostic/therapeutic errors. The themes associated with hospital type (referral, urban, regional/rural) were also similar. Although theme analysis is important, some individual cases were particularly instructive. CONCLUSION: The NCIS data theme analysis identifies important high-risk patients and presenting complaints. These should be incorporated into emergency physician training. EDs should review the coronial recommendations to ensure that, where possible, they have been adopted.

Medication use in children and adolescents treated in the community for bipolar disorder.

We assessed the use of mood stabilizers, stimulants, antipsychotic medication, and selective serotonin reuptake inhibitors in children being treated in the community for bipolar disorder (BPD). One hundred eleven patients were screened via parent phone interview for possible inclusion in a phenomenological study of BPD. Data were obtained on the patients' medication trials and side effects. The results of the study indicated that children and adolescents who carry a diagnosis of BPD are treated with a mean of 3.40 +/- 1.48 medications and have had a mean of 6.32 +/- 3.67 trials of psychotropic medication in the past. Ninety-eight percent have had a trial of a mood stabilizer or anticonvulsant, with the most common being valproate (79%), lithium (51%), and gabapentin (29%).