Genome-wide association studies of Down syndrome associated congenital heart defects.

Congenital heart defects (CHDs) are the most common structural birth defect and are present in 40-50% of children born with Down syndrome (DS). To characterize the genetic architecture of DS-associated CHD, we sequenced genomes of a multiethnic group of children with DS and a CHD (n=886: atrioventricular septal defects (AVSD), n=438; atrial septal defects (ASD), n=122; ventricular septal defects (VSD), n=170; other types of CHD, n=156) and DS with a structurally normal heart (DS+NH, n=572). We performed four GWAS for common variants (MAF>0.05) comparing DS with CHD, stratified by CHD-subtype, to DS+NH controls. Although no SNP achieved genome-wide significance, multiple loci in each analysis achieved suggestive significance (p<2×10-6). Of these, the 1p35.1 locus (near RBBP4) was specifically associated with ASD risk and the 5q35.2 locus (near MSX2) was associated with any type of CHD. Each of the suggestive loci contained one or more plausible candidate genes expressed in the developing heart. While no SNP replicated (p<2×10-6) in an independent cohort of DS+CHD (DS+CHD: n=229; DS+NH: n=197), most SNPs that were suggestive in our GWASs remained suggestive when meta-analyzed with the GWASs from the replication cohort. These results build on previous work to identify genetic modifiers of DS-associated CHD.

Combined Whole Eye and Face Transplant: Microsurgical Strategy and 1-Year Clinical Course.

Importance: Catastrophic facial injury with globe loss remains a formidable clinical problem with no previous reports of reconstruction by whole eye or combined whole eye and facial transplant. Objective: To develop a microsurgical strategy for combined whole eye and facial transplant and describe the clinical findings during the first year following transplant. Design, Setting, and Participant: A 46-year-old man who sustained a high-voltage electrical injury with catastrophic tissue loss to his face and left globe underwent combined whole eye and face transplant using personalized surgical devices and a novel microsurgical strategy at a specialized center for vascularized composite allotransplantation. Main Outcomes and Measures: Reperfusion and viability of the whole eye and facial allografts, retinal function, and incidence of acute rejection. Results: The patient underwent a combined whole eye and face transplant from an immunologically compatible donor with primary optic nerve coaptation and conventional postoperative immunosuppression. Globe and retinal perfusion were maintained throughout the immediate postoperative period, evidenced by fluorescein angiography. Optical coherence tomography demonstrated atrophy of inner retinal layers and attenuation and disruption of the ellipsoid zone. Serial electroretinography confirmed retinal responses to light in the transplanted eye. Using structural and functional magnetic resonance imaging, the integrity of the transplanted visual pathways and potential occipital cortical response to light stimulation of the transplanted eye was demonstrated. At 1 year post transplant (postoperative day 366), there was no perception of light in the transplanted eye. Conclusions and Relevance: This is the first report of whole eye transplant combined with facial transplant, demonstrating allograft survival including rejection-free graft survival and electroretinographic measurements indicating retinal response to light stimuli. These data highlight the potential for clinical allotransplantation for globe loss.

The BabySeq Project: A clinical trial of genome sequencing in a diverse cohort of infants.

Efforts to implement and evaluate genome sequencing (GS) as a screening tool for newborns and infants are expanding worldwide. The first iteration of the BabySeq Project (2015-2019), a randomized controlled trial of newborn sequencing, produced novel evidence on medical, behavioral, and economic outcomes. The second iteration of BabySeq, which began participant recruitment in January 2023, examines GS outcomes in a larger, more diverse cohort of more than 500 infants up to one year of age recruited from pediatric clinics at several sites across the United States. The trial aims for families who self-identify as Black/African American or Hispanic/Latino to make up more than 50% of final enrollment, and key aspects of the trial design were co-developed with a community advisory board. All enrolled families receive genetic counseling and a family history report. Half of enrolled infants are randomized to receive GS with comprehensive interpretation of pathogenic and likely pathogenic variants in more than 4,300 genes associated with childhood-onset and actionable adult-onset conditions, as well as larger-scale chromosomal copy number variants classified as pathogenic or likely pathogenic. GS result reports include variants associated with disease (Mendelian disease risks) and carrier status of autosomal-recessive and X-linked disorders. Investigators evaluate the utility and impacts of implementing a GS screening program in a diverse cohort of infants using medical record review and longitudinal parent surveys. In this perspective, we describe the rationale for the second iteration of the BabySeq Project, the outcomes being assessed, and the key decisions collaboratively made by the study team and community advisory board.

Sudden Death in Pediatric Patient With Dilated Cardiomyopathy Due to Founder Variant in NKX2-5: Case Report.

Background: The NKX2-5 gene encodes a transcription factor that plays a role in atrioventricular nodal and myocardial development. Pathogenic variants of NKX2-5 are associated with congenital heart disease and sudden cardiac death. The missense variant in this case is one of the more common ones in Northern Europe and has high penetrance in familial cases. To our knowledge, this is the youngest person who died due to this variant. Case summary: This was a healthy, asymptomatic 14-year-old male with well-managed mild congenital dilated cardiomyopathy who died unexpectedly in his home. Postmortem examination revealed the NKX2-5 pathogenic missense variant, p.Phe145Leu, as the only explicable cause of death. Discussion: We propose that immediate family members of those who die suddenly due to NKX2-5 disease undergo genetic counseling and longitudinal screening to include this gene, as pathogenic variants in the NKX2-5 gene may manifest in a time-dependent manner.

Aortic Root Dilation and Genotype Associations in Phelan-McDermid Syndrome.

Phelan-McDermid syndrome (PMS) is a rare genetic neurodevelopmental disorder that results from the loss of one functional copy of the SHANK3 gene. While many clinical features of PMS are well-understood, there is currently limited literature on cardiovascular abnormalities in PMS. This report aims to evaluate the prevalence of aortic root dilation (ARD) among individuals with PMS and to understand if underlying genetic variation relates to risk for ARD. We present findings from 59 participants collected from a multisite observational study evaluating the phenotype and natural history of PMS. Individual echocardiographic and genetic reports were analyzed for aortic root measurements and genetic variant data, respectively. Our a priori hypothesis was that participants with chromosome 22 deletions with hg19 start coordinates on or before 49,900,000 (larger deletions) would have more instances of ARD than participants with deletion start coordinates after 49,900,000 (smaller deletions). Eight participants (14%) had ARD, and its presence was statistically significantly associated with large deletions (p = 0.047). Relatedly, participants with ARD had significantly more genes deleted on chromosome 22 than participants without ARD (p = 0.013). These results could aid in the identification of individuals with PMS who are at higher risk for ARD.

Trisomy 21 and Congenital Heart Disease: Impact on Health and Functional Outcomes From Birth Through Adolescence: A Scientific Statement From the American Heart Association.

Due to improvements in recognition and management of their multisystem disease, the long-term survival of infants, children, and adolescents with trisomy 21 and congenital heart disease now matches children with congenital heart disease and no genetic condition in many scenarios. Although this improved survival is a triumph, individuals with trisomy 21 and congenital heart disease have unique and complex care needs in the domains of physical, developmental, and psychosocial health, which affect functional status and quality of life. Pulmonary hypertension and single ventricle heart disease are 2 known cardiovascular conditions that reduce life expectancy in individuals with trisomy 21. Multisystem involvement with respiratory, endocrine, gastrointestinal, hematological, neurological, and sensory systems can interact with cardiovascular health concerns to amplify adverse effects. Neurodevelopmental, psychological, and functional challenges can also affect quality of life. A highly coordinated interdisciplinary care team model, or medical home, can help address these complex and interactive conditions from infancy through the transition to adult care settings. The purpose of this Scientific Statement is to identify ongoing cardiovascular and multisystem, developmental, and psychosocial health concerns for children with trisomy 21 and congenital heart disease from birth through adolescence and to provide a framework for monitoring and management to optimize quality of life and functional status.

An Update on the Survival of the First 50 Face Transplants Worldwide.

Importance: Since 2005, a total of 50 face transplants have been reported from 18 centers in 11 countries. The overall survival of the grafts has not yet been established. Objective: To assess the survival of the face transplant grafts and evaluate factors potentially influencing it. Design, Setting, and Participants: Data on all the transplants included in this multicenter cohort study were collected at participating transplant centers for updated nonpublished data, supplemented with literature review for nonparticipating centers. Data from 2005 until September 2023, were included. Data were analyzed from November 11, 2005, through September 18, 2023. Patients included the first 50 patients in the world to have received a face transplant. Exposure: Face transplant graft. Main Outcomes and Measures: The primary outcome was the overall survival of the face transplant graft, defined as either transplant loss or patient death. The secondary outcome was the number of acute rejection episodes per year. Results: The 50 transplants were performed on 39 men (81%) and 9 women (19%) with a median age of 35 (range, 19-68) years at the time of the transplant. The median follow-up time was 8.9 (range, 0.2-16.7) years. During the follow-up, 6 transplants were lost with 2 patients retransplanted. There were 10 patients who died, 2 of whom had lost a transplant. The 5- and 10-year survival of the transplants was 85% (SD, 5%) and 74% (SD, 7%), respectively. The sequential number of the transplant in the world was a significant predictor of survival (hazard ratio, 95; 95% CI, 90-100; P < 05). The median number of acute rejection episodes per year was 1.2 (range, 0-5.3) for the transplants that were lost and 0.7 (range, 0-4.6) for the transplants that survived. No correlation with patient and transplant variables was detected for either the transplant survival or the number of rejection episodes. Conclusions and Relevance: In this study, the overall survival of the face transplants is encouraging. These data suggest that the acceptable long-term survival of face transplants makes them a reconstructive option for extensive facial defects.

Molecular Signature Associated With Acute Rejection in Vascularized Composite Allotransplantation.

Background: A deeper understanding of acute rejection in vascularized composite allotransplantation is paramount for expanding its utility and longevity. There remains a need to develop more precise and accurate tools for diagnosis and prognosis of these allografts, as well as alternatives to traditional immunosuppressive regimens. Methods: Twenty-seven skin biopsies collected from 3 vascularized composite allotransplantation recipients, consisting of face and hand transplants, were evaluated by histology, immunohistochemistry staining, and gene expression profiling. Results: Biopsies with clinical signs and symptoms of rejection, irrespective of histopathological grading, were significantly enriched for genes contributing to the adaptive immune response, innate immune response, and lymphocyte activation. Inflammation episodes exhibited significant fold change correlations between the face and hands, as well as across patients. Immune checkpoint genes were upregulated during periods of inflammation that necessitated treatment. A gene signature consisting of CCL5, CD8A, KLRK1, and IFNγ significantly predicted inflammation specific to vascularized composite allografts that required therapeutic intervention. Conclusions: The mechanism of vascularized composite allograft-specific inflammation and rejection appears to be conserved across different patients and skin on different anatomical sites. A concise gene signature can be utilized to ascertain graft status along with a continuous scale, providing valuable diagnostic and prognostic information to supplement current gold standards of graft evaluation.

Outcomes with distal transradial access in patients with advanced chronic kidney disease.

Radial artery occlusion (RAO), a complication of transradial access, has an incidence of 4.0% to 9.1% in patients with advanced chronic kidney disease (CKD) and may preclude its use creation of arteriovenous fistula. Distal transradial access (dTRA) has lower rates of RAO compared with TRA, but prior studies excluded patients with advanced CKD. This was a single center study of patients with advanced CKD who underwent coronary procedures with dTRA from January 1, 2019 to May 12, 2022 who were retrospectively evaluated for radial artery patency in follow-up with reverse Barbeau testing or repeat access of the artery. Of 71 patients, 66% were on hemodialysis and the remainder had CKD 3 to 5. Access was ultrasound-guided, and all received adequate spasmolytic therapy and patent hemostasis. Proximal radial arteries were patent in 100% of the patients at follow-up. Our data suggest that dTRA is safe for patients with advanced CKD and preserves radial artery patency.

Identifying novel data-driven subgroups in congenital heart disease using multi-modal measures of brain structure.

Individuals with congenital heart disease (CHD) have an increased risk of neurodevelopmental impairments. Given the hypothesized complexity linking genomics, atypical brain structure, cardiac diagnoses and their management, and neurodevelopmental outcomes, unsupervised methods may provide unique insight into neurodevelopmental variability in CHD. Using data from the Pediatric Cardiac Genomics Consortium Brain and Genes study, we identified data-driven subgroups of individuals with CHD from measures of brain structure. Using structural magnetic resonance imaging (MRI; N = 93; cortical thickness, cortical volume, and subcortical volume), we identified subgroups that differed primarily on cardiac anatomic lesion and language ability. In contrast, using diffusion MRI (N = 88; white matter connectivity strength), we identified subgroups that were characterized by differences in associations with rare genetic variants and visual-motor function. This work provides insight into the differential impacts of cardiac lesions and genomic variation on brain growth and architecture in patients with CHD, with potentially distinct effects on neurodevelopmental outcomes.

Evaluating parental personal utility of pediatric genetic and genomic testing in a diverse, multilingual population.

There is increasing evidence of the clinical utility of genetic and genomic testing (GT); however, factors influencing personal utility of GT, especially in diverse, multilingual populations, remain unclear. We explored these factors in a diverse cohort of parents/guardians (participants) whose children received clinical GT through the NYCKidSeq program. A total of 847 participants completed surveys at baseline, post-results disclosure, and 6 months (6m) post-results. The largest population groups were Hispanic/Latino(a) (48%), White/European American (24%), and Black/African American (16%). Personal utility was assessed using the Personal Utility (PrU) scale, adapted for pediatric populations and included on the surveys. Three PrU subscales were identified using factor analysis: practical, educational, and parental psychological utility. Overall personal utility summary score and the three subscales significantly decreased after receiving results and over time. Hispanic/Latino(a) participants identified greater overall personal utility than European American and African American participants at all time points (p < 0.001) as did participants whose children received positive/likely positive results compared with those with negative and uncertain results (post-results: p < 0.001 and p < 0.001; 6m post-results: p = 0.002 and p < 0.001, respectively). Post-results, higher subscale scores were associated with lower education levels (practical, parental psychological: p ≤ 0.02) and higher levels of trust in the healthcare system (practical, parental psychological: p ≤ 0.04). These findings help to understand the perspectives of diverse parents/guardians, which is critical to tailoring pre- and post-test counseling across a variety of populations and clinical settings.

Genome Sequencing is Critical for Forecasting Outcomes following Congenital Cardiac Surgery.

While genome sequencing has transformed medicine by elucidating the genetic underpinnings of both rare and common complex disorders, its utility to predict clinical outcomes remains understudied. Here, we used artificial intelligence (AI) technologies to explore the predictive value of genome sequencing in forecasting clinical outcomes following surgery for congenital heart defects (CHD). We report results for a cohort of 2,253 CHD patients from the Pediatric Cardiac Genomics Consortium with a broad range of complex heart defects, pre- and post-operative clinical variables and exome sequencing. Damaging genotypes in chromatin-modifying and cilia-related genes were associated with an elevated risk of adverse post-operative outcomes, including mortality, cardiac arrest and prolonged mechanical ventilation. The impact of damaging genotypes was further amplified in the context of specific CHD phenotypes, surgical complexity and extra-cardiac anomalies. The absence of a damaging genotype in chromatin-modifying and cilia-related genes was also informative, reducing the risk for adverse postoperative outcomes. Thus, genome sequencing enriches the ability to forecast outcomes following congenital cardiac surgery.

Cardiac genetic test yields and genotype-phenotype correlations from large cohort investigated by medical examiner's office.

BACKGROUND: Few reports describe the yield of postmortem genetic testing from medical examiners' offices or correlate genetic test results with autopsy-confirmed phenotypes from a large cohort. OBJECTIVES: To report results from cardiomyopathy- and cardiac arrhythmia-associated genetic testing in conjunction with autopsy findings of cases investigated at the United States' largest medical examiner office. METHODS: Postmortem cases tested from 2015 to 2022 with a cardiomyopathy- and cardiac arrhythmia-associated gene panel were reviewed. American College of Medical Genetics and Genomics/Association for Molecular Pathology guidelines were used to classify variant pathogenicity. Correlations of pathogenic/likely pathogenic variants (P/LPVs) with cardiac pathology were evaluated. RESULTS: The cohort included 1107 decedents of diverse ages and ethnicities. P/LPVs were detected in 87 (7.9%) cases, with 73 and 14 variants in cardiomyopathy and cardiac arrhythmia genes, respectively. Variants of uncertain significance were detected in 437 (39.5%) cases. The diagnostic yield (percentage of P/LPV) in decedents with cardiomyopathy (26.1%) was significantly higher than those without (P<.0001). The diagnostic yield was significantly lower in infants (0.7%) than older age groups (ranging from 1 to 74 years old, 5.7%-25.9%), which had no statistical difference between their yields. The diagnostic yields by cardiac autopsy findings were 54.0% for hypertrophic cardiomyopathy, 47.1% for arrhythmogenic cardiomyopathy, 20.0% for myocardial fibrosis, 19.0% for dilated cardiomyopathy, and 11.3% for myocarditis. Most P/LPVs were in MYBPC3, TTN, PKP2, SCN5A, MYH7, and FLNC. Ten P/LPVs were novel. CONCLUSIONS: Our results support the importance of performing postmortem genetic testing on decedents of all ages with cardiomyopathy, cardiac lesions insufficient to diagnosis a specific cardiomyopathy (e.g., myocardial fibrosis), and myocarditis. Combined postmortem cardiac examination and genetic analysis are advantageous in accurately determining the underlying cause of death and informing effective clinical care of family members.

Physician and informal care use explained by the Pediatric Quality of Life Inventory (PedsQL) in children with suspected genetic disorders.

PURPOSE: To examine associations between Pediatric Quality of Life Inventory (PedsQL) 4.0 Generic Core Scales and PedsQL Infant Scales with formal health care resource utilization (HCRU) and informal caregiver burden. METHODS: We studied a pediatric cohort of 837 patients (median age: 8.4 years) with suspected genetic disorders enrolled January 2019 through July 2021 in the NYCKidSeq program for diagnostic sequencing. Using linked ~ nine-month longitudinal survey and physician claims data collected through May 2022, we modeled the association between baseline PedsQL scores and post-baseline HCRU (median follow-up: 21.1 months) and informal care. We also assessed the longitudinal change in PedsQL scores with physician services using linear mixed-effects models. RESULTS: Lower PedsQL total and physical health scores were independently associated with increases in 18-month physician services, encounters, and weekly informal care. Comparing low vs. median total scores, increases were 10.6 services (95% CI: 1.0-24.6), 3.3 encounters (95% CI: 0.5-6.8), and $668 (95% CI: $350-965), respectively. For the psychosocial domain, higher scores were associated with decreased informal care. Based on adjusted linear mixed-effects modeling, every additional ten physician services was associated with diminished improvement in longitudinal PedsQL total score trajectories by 1.1 point (95% confidence interval: 0.6-1.6) on average. Similar trends were observed in the physical and psychosocial domains. CONCLUSION: PedsQL scores were independently associated with higher utilization of physician services and informal care. Moreover, longitudinal trajectories of PedsQL scores became less favorable with increased physician services. Adding PedsQL survey instruments to conventional measures for improved risk stratification should be evaluated in further research.

The Pediatric Quality of Life Inventory (PedsQL) is widely used to measure health-related quality of life in pediatric patients; however, few studies have examined whether the PedsQL is indicative of longitudinal outcomes of morbidity and health care needs. This study captures associations between PedsQL scores with utilization of physician and informal care in children with suspected genetic disorders. We demonstrate that lower PedsQL total and physical health scores are independently associated with greater utilization of physician services and informal care. Moreover, longitudinal trajectories of PedsQL scores become less favorable with increased physician services. Results can inform future applications of PedsQL instruments.

Automated Identification of Germline de novo Mutations in Family Trios: A Consensus-Based Informatic Approach.

Accurate identification of germline de novo variants (DNVs) remains a challenging problem despite rapid advances in sequencing technologies as well as methods for the analysis of the data they generate, with putative solutions often involving ad hoc filters and visual inspection of identified variants. Here, we present a purely informatic method for the identification of DNVs by analyzing short-read genome sequencing data from proband-parent trios. Our method evaluates variant calls generated by three genome sequence analysis pipelines utilizing different algorithms-GATK HaplotypeCaller, DeepTrio and Velsera GRAF-exploring the assumption that a requirement of consensus can serve as an effective filter for high-quality DNVs. We assessed the efficacy of our method by testing DNVs identified using a previously established, highly accurate classification procedure that partially relied on manual inspection and used Sanger sequencing to validate a DNV subset comprising less confident calls. The results show that our method is highly precise and that applying a force-calling procedure to putative variants further removes false-positive calls, increasing precision of the workflow to 99.6%. Our method also identified novel DNVs, 87% of which were validated, indicating it offers a higher recall rate without compromising accuracy. We have implemented this method as an automated bioinformatics workflow suitable for large-scale analyses without need for manual intervention.

NPSV-deep: a deep learning method for genotyping structural variants in short read genome sequencing data.

MOTIVATION: Structural variants (SVs) play a causal role in numerous diseases but can be difficult to detect and accurately genotype (determine zygosity) with short-read genome sequencing data (SRS). Improving SV genotyping accuracy in SRS data, particularly for the many SVs first detected with long-read sequencing, will improve our understanding of genetic variation. RESULTS: NPSV-deep is a deep learning-based approach for genotyping previously reported insertion and deletion SVs that recasts this task as an image similarity problem. NPSV-deep predicts the SV genotype based on the similarity between pileup images generated from the actual SRS data and matching SRS simulations. We show that NPSV-deep consistently matches or improves upon the state-of-the-art for SV genotyping accuracy across different SV call sets, samples and variant types, including a 25% reduction in genotyping errors for the Genome-in-a-Bottle (GIAB) high-confidence SVs. NPSV-deep is not limited to the SVs as described; it improves deletion genotyping concordance a further 1.5 percentage points for GIAB SVs (92%) by automatically correcting imprecise/incorrectly described SVs. AVAILABILITY AND IMPLEMENTATION: Python/C++ source code and pre-trained models freely available at https://github.com/mlinderm/npsv2.

Diagnostic yield after next-generation sequencing in pediatric cardiovascular disease.

Genetic testing with exome sequencing and genome sequencing is increasingly offered to infants and children with cardiovascular diseases. However, the rates of positive diagnoses after genetic testing within the different categories of cardiac disease and phenotypic subtypes of congenital heart disease (CHD) have been little studied. We report the diagnostic yield after next-generation sequencing in 500 patients with CHD from diverse population subgroups that were enrolled at three different sites in the Clinical Sequencing Evidence-Generating Research consortium. Patients were ascertained due to a primary cardiovascular issue comprising arrhythmia, cardiomyopathy, and/or CHD, and corresponding human phenotype ontology terms were selected to describe the cardiac and extracardiac findings. We examined the diagnostic yield for patients with arrhythmia, cardiomyopathy, and/or CHD and phenotypic subtypes of CHD comprising conotruncal defects, heterotaxy, left ventricular outflow tract obstruction, septal defects, and "other" heart defects. We found a significant increase in the frequency of positive findings for patients who underwent genome sequencing compared to exome sequencing and for syndromic cardiac defects compared to isolated cardiac defects. We also found significantly higher diagnostic rates for patients who presented with isolated cardiomyopathy compared to isolated CHD. For patients with syndromic presentations who underwent genome sequencing, there were significant differences in the numbers of positive diagnoses for phenotypic subcategories of CHD, ranging from 31.7% for septal defects to 60% for "other". Despite variation in the diagnostic yield at each site, our results support genetic testing in pediatric patients with syndromic and isolated cardiovascular issues and in all subtypes of CHD.

Functional dissection of human cardiac enhancers and noncoding de novo variants in congenital heart disease.

Rare coding mutations cause ∼45% of congenital heart disease (CHD). Noncoding mutations that perturb cis-regulatory elements (CREs) likely contribute to the remaining cases, but their identification has been problematic. Using a lentiviral massively parallel reporter assay (lentiMPRA) in human induced pluripotent stem cell-derived cardiomyocytes (iPSC-CMs), we functionally evaluated 6,590 noncoding de novo variants (ncDNVs) prioritized from the whole-genome sequencing of 750 CHD trios. A total of 403 ncDNVs substantially affected cardiac CRE activity. A majority increased enhancer activity, often at regions with undetectable reference sequence activity. Of ten DNVs tested by introduction into their native genomic context, four altered the expression of neighboring genes and iPSC-CM transcriptional state. To prioritize future DNVs for functional testing, we used the MPRA data to develop a regression model, EpiCard. Analysis of an independent CHD cohort by EpiCard found enrichment of DNVs. Together, we developed a scalable system to measure the effect of ncDNVs on CRE activity and deployed it to systematically assess the contribution of ncDNVs to CHD.

HRAS-Mutant Cardiomyocyte Model of Multifocal Atrial Tachycardia.

BACKGROUND: Germline HRAS gain-of-function pathogenic variants cause Costello syndrome (CS). During early childhood, 50% of patients develop multifocal atrial tachycardia, a treatment-resistant tachyarrhythmia of unknown pathogenesis. This study investigated how overactive HRAS activity triggers arrhythmogenesis in atrial-like cardiomyocytes (ACMs) derived from human-induced pluripotent stem cells bearing CS-associated HRAS variants. METHODS: HRAS Gly12 mutations were introduced into a human-induced pluripotent stem cells-ACM reporter line. Human-induced pluripotent stem cells were generated from patients with CS exhibiting tachyarrhythmia. Calcium transients and action potentials were assessed in induced pluripotent stem cell-derived ACMs. Automated patch clamping assessed funny currents. HCN inhibitors targeted pacemaker-like activity in mutant ACMs. Transcriptomic data were analyzed via differential gene expression and gene ontology. Immunoblotting evaluated protein expression associated with calcium handling and pacemaker-nodal expression. RESULTS: ACMs harboring HRAS variants displayed higher beating rates compared with healthy controls. The hyperpolarization activated cyclic nucleotide gated potassium channel inhibitor ivabradine and the Nav1.5 blocker flecainide significantly decreased beating rates in mutant ACMs, whereas voltage-gated calcium channel 1.2 blocker verapamil attenuated their irregularity. Electrophysiological assessment revealed an increased number of pacemaker-like cells with elevated funny current densities among mutant ACMs. Mutant ACMs demonstrated elevated gene expression (ie, ISL1, TBX3, TBX18) related to intracellular calcium homeostasis, heart rate, RAS signaling, and induction of pacemaker-nodal-like transcriptional programming. Immunoblotting confirmed increased protein levels for genes of interest and suppressed MAPK (mitogen-activated protein kinase) activity in mutant ACMs. CONCLUSIONS: CS-associated gain-of-function HRASG12 mutations in induced pluripotent stem cells-derived ACMs trigger transcriptional changes associated with enhanced automaticity and arrhythmic activity consistent with multifocal atrial tachycardia. This is the first human-induced pluripotent stem cell model establishing the mechanistic basis for multifocal atrial tachycardia in CS.

Banff 2022 Vascularized Composite Allotransplantation Meeting Report: Diagnostic criteria for vascular changes.

As more data become available, the Banff 2007 working classification of skin-containing vascularized composite allograft (VCA) pathology is expected to evolve and develop. This report represents the Banff VCA Working Group's consensus on the first revision of the 2007 scoring system. Prior to the 2022 Banff-CanXadian Society of Transplantation Joint Meeting, 83 clinicians and/or researchers were invited to a virtual meeting to discuss whether the 2007 Banff VCA system called for a revision. Unanimously, it was determined that the vascular changes were to be included in the first revision. Subsequently, 2 international online surveys, each followed by virtual discussions, were launched. The goals were (1) to identify which changes define severe rejection, (2) to grade their importance in the evaluation of severe rejection, and (3) to identify emerging criteria to diagnose rejection. A final hybrid (in-person and virtual) discussion at the Banff/Canadian Society of Transplantation Joint Meeting finalized the terminology, the definition, a scoring system, and a reporting system of the vascular changes. This proposal represents an international consensus on this topic and establishes the first revision of the Banff 2007 working classification of skin-containing vascularized composite allograft pathology.