Myocarditis and Pericarditis Following mRNA COVID-19 Vaccination: 2024 Status and Management Update.

In 2021, the Canadian Journal of Cardiology published a "Practical Clinical Practice Update" to address emerging concerns about myocarditis and pericarditis following mRNA COVID-19 vaccines.1 Since publication, researchers have underscored the importance of standardizing diagnostic criteria and evaluation of incident cases. In this brief review, we summarize new evidence about the epidemiology and outcomes of patients with mRNA COVID-19 post-vaccine myocarditis and pericarditis. We also provide consensus guidance for evaluation, management, and follow-up. Finally, we identify persistent knowledge gaps that inform the National Active Surveillance Study of Myocarditis and/or Pericarditis following mRNA COVID-19 Vaccination (MYCOVACC) and areas for future research.

Spliceosome malfunction causes neurodevelopmental disorders with overlapping features.

Pre-mRNA splicing is a highly coordinated process. While its dysregulation has been linked to neurological deficits, our understanding of the underlying molecular and cellular mechanisms remains limited. We implicated pathogenic variants in U2AF2 and PRPF19, encoding spliceosome subunits in neurodevelopmental disorders (NDDs), by identifying 46 unrelated individuals with 23 de novo U2AF2 missense variants (including 7 recurrent variants in 30 individuals) and 6 individuals with de novo PRPF19 variants. Eight U2AF2 variants dysregulated splicing of a model substrate. Neuritogenesis was reduced in human neurons differentiated from human pluripotent stem cells carrying two U2AF2 hyper-recurrent variants. Neural loss of function (LoF) of the Drosophila orthologs U2af50 and Prp19 led to lethality, abnormal mushroom body (MB) patterning, and social deficits, which were differentially rescued by wild-type and mutant U2AF2 or PRPF19. Transcriptome profiling revealed splicing substrates or effectors (including Rbfox1, a third splicing factor), which rescued MB defects in U2af50-deficient flies. Upon reanalysis of negative clinical exomes followed by data sharing, we further identified 6 patients with NDD who carried RBFOX1 missense variants which, by in vitro testing, showed LoF. Our study implicates 3 splicing factors as NDD-causative genes and establishes a genetic network with hierarchy underlying human brain development and function.

Report of two cases of Schaaf-Yang syndrome: Same genotype and different phenotype.

We report two, genotypically identical but phenotypically distinct cases of Schaaf-Yang syndrome and propose the early use of Genome Sequencing in patients with nonspecific presentations to facilitate the early diagnosis of children with rare genetic diseases and improve overall health care outcomes.

Fc-competent multispecific PDL-1/TIGIT/LAG-3 antibodies potentiate superior anti-tumor T cell response.

The landscape of current cancer immunotherapy is dominated by antibodies targeting PD-1/PD-L1 and CTLA-4 that have transformed cancer therapy, yet their efficacy is limited by primary and acquired resistance. The blockade of additional immune checkpoints, especially TIGIT and LAG-3, has been extensively explored, but so far only a LAG-3 antibody has been approved for combination with nivolumab to treat unresectable or metastatic melanoma. Here we report the development of a PDL1 × TIGIT bi-specific antibody (bsAb) GB265, a PDL1 × LAG3 bsAb GB266, and a PDL1 × TIGIT × LAG3 tri-specific antibody (tsAb) GB266T, all with intact Fc function. In in vitro cell-based assays, these antibodies promote greater T cell expansion and tumor cell killing than benchmark antibodies and antibody combinations in an Fc-dependent manner, likely by facilitating T cell interactions (bridging) with cancer cells and monocytes, in addition to blocking immune checkpoints. In animal models, GB265 and GB266T antibodies outperformed benchmarks in tumor suppression. This study demonstrates the potential of a new generation of multispecific checkpoint inhibitors to overcome resistance to current monospecific checkpoint antibodies or their combinations for the treatment of human cancers.

Rapid Whole Genome Sequencing for Diagnosis of Single Locus Genetic Diseases in Critically Ill Children.

Upon admission to intensive care units (ICU), the differential diagnosis of almost all infants with diseases of unclear etiology includes single locus genetic diseases. Rapid whole genome sequencing (rWGS), including sample preparation, short-read sequencing-by-synthesis, informatics pipelining, and semiautomated interpretation, can now identify nucleotide and structural variants associated with most genetic diseases with robust analytic and diagnostic performance in as little as 13.5 h. Early diagnosis of genetic diseases transforms medical and surgical management of infants in ICUs, minimizing both the duration of empiric treatment and the delay to start of specific treatment. Both positive and negative rWGS tests have clinical utility and can improve outcomes. Since first described 10 years ago, rWGS has evolved considerably. Here we describe our current methods for routine diagnostic testing for genetic diseases by rWGS in as little as 18 h.

Reclassification of the Etiology of Infant Mortality With Whole-Genome Sequencing.

Importance: Understanding the causes of infant mortality shapes public health, surveillance, and research investments. However, the association of single-locus (mendelian) genetic diseases with infant mortality is poorly understood. Objective: To determine the association of genetic diseases with infant mortality. Design, Setting, and Participants: This cohort study was conducted at a large pediatric hospital system in San Diego County (California) and included 546 infants (112 infant deaths [20.5%] and 434 infants [79.5%] with acute illness who survived; age, 0 to 1 year) who underwent diagnostic whole-genome sequencing (WGS) between January 2015 and December 2020. Data analysis was conducted between 2015 and 2022. Exposure: Infants underwent WGS either premortem or postmortem with semiautomated phenotyping and diagnostic interpretation. Main Outcomes and Measures: Proportion of infant deaths associated with single-locus genetic diseases. Results: Among 112 infant deaths (54 girls [48.2%]; 8 [7.1%] African American or Black, 1 [0.9%] American Indian or Alaska Native, 8 [7.1%] Asian, 48 [42.9%] Hispanic, 1 [0.9%] Native Hawaiian or Pacific Islander, and 34 [30.4%] White infants) in San Diego County between 2015 and 2020, single-locus genetic diseases were the most common identifiable cause of infant mortality, with 47 genetic diseases identified in 46 infants (41%). Thirty-nine (83%) of these diseases had been previously reported to be associated with childhood mortality. Twenty-eight death certificates (62%) for 45 of the 46 infants did not mention a genetic etiology. Treatments that can improve outcomes were available for 14 (30%) of the genetic diseases. In 5 of 7 infants in whom genetic diseases were identified postmortem, death might have been avoided had rapid, diagnostic WGS been performed at time of symptom onset or regional intensive care unit admission. Conclusions and Relevance: In this cohort study of 112 infant deaths, the association of genetic diseases with infant mortality was higher than previously recognized. Strategies to increase neonatal diagnosis of genetic diseases and immediately implement treatment may decrease infant mortality. Additional study is required to explore the generalizability of these findings and measure reduction in infant mortality.

Autosomal recessive LRP1-related syndrome featuring cardiopulmonary dysfunction, bone dysmorphology, and corneal clouding.

We provide the first study of two siblings with a novel autosomal recessive LRP1-related syndrome identified by rapid genome sequencing and overlapping multiple genetic models. The patients presented with respiratory distress, congenital heart defects, hypotonia, dysmorphology, and unique findings, including corneal clouding and ascites. Both siblings had compound heterozygous damaging variants, c.11420G > C (p.Cys3807Ser) and c.12407T > G (p.Val4136Gly) in LRP1, in which segregation analysis helped dismiss additional variants of interest. LRP1 analysis using multiple human/mouse data sets reveals a correlation to patient phenotypes of Peters plus syndrome with additional severe cardiomyopathy and blood vessel development complications linked to neural crest cells.

A novel IgG Fc by computer-aided design enhances heavy-chain heterodimerization in bi- or trispecific antibodies.

The classical `knob-into-holes' (KIH) strategy (knob(T366Y)/hole (Y407T)) has successfully enhanced the heterodimerization of a bispecific antibody (BsAb) resulting in heterodimer formation up to 92% of protein A (ProA)-purified protein pool. However, it does not show high efficiency for every BsAb. KIH was initially applied to a CD20/CD3 BsAb. After in silico modeling, two additional new mutations, S354Y in knob-heavy chain (HC) and Q347E in hole-HC, together with KIH named `ETYY', were introduced in the Fc. The CD20/CD3 BsAb hybrid only represented ~ 50% of the ProA-purified protein pool when KIH was applied. With ETYY, the percentage of CD20/CD3 hybrid increased to 93.8%. CD20/CD3-v4b (containing ETYY) retains the original activity of the BsAb at both Fab and Fc regions, and also shows good developability. These results indicate that the computer-aided novel ETYY design has the potential to improve the development of next-generation BsAbs with higher yields and simpler purification.

A genome sequencing system for universal newborn screening, diagnosis, and precision medicine for severe genetic diseases.

Newborn screening (NBS) dramatically improves outcomes in severe childhood disorders by treatment before symptom onset. In many genetic diseases, however, outcomes remain poor because NBS has lagged behind drug development. Rapid whole-genome sequencing (rWGS) is attractive for comprehensive NBS because it concomitantly examines almost all genetic diseases and is gaining acceptance for genetic disease diagnosis in ill newborns. We describe prototypic methods for scalable, parentally consented, feedback-informed NBS and diagnosis of genetic diseases by rWGS and virtual, acute management guidance (NBS-rWGS). Using established criteria and the Delphi method, we reviewed 457 genetic diseases for NBS-rWGS, retaining 388 (85%) with effective treatments. Simulated NBS-rWGS in 454,707 UK Biobank subjects with 29,865 pathogenic or likely pathogenic variants associated with 388 disorders had a true negative rate (specificity) of 99.7% following root cause analysis. In 2,208 critically ill children with suspected genetic disorders and 2,168 of their parents, simulated NBS-rWGS for 388 disorders identified 104 (87%) of 119 diagnoses previously made by rWGS and 15 findings not previously reported (NBS-rWGS negative predictive value 99.6%, true positive rate [sensitivity] 88.8%). Retrospective NBS-rWGS diagnosed 15 children with disorders that had been undetected by conventional NBS. In 43 of the 104 children, had NBS-rWGS-based interventions been started on day of life 5, the Delphi consensus was that symptoms could have been avoided completely in seven critically ill children, mostly in 21, and partially in 13. We invite groups worldwide to refine these NBS-rWGS conditions and join us to prospectively examine clinical utility and cost effectiveness.

An automated 13.5 hour system for scalable diagnosis and acute management guidance for genetic diseases.

While many genetic diseases have effective treatments, they frequently progress rapidly to severe morbidity or mortality if those treatments are not implemented immediately. Since front-line physicians frequently lack familiarity with these diseases, timely molecular diagnosis may not improve outcomes. Herein we describe Genome-to-Treatment, an automated, virtual system for genetic disease diagnosis and acute management guidance. Diagnosis is achieved in 13.5 h by expedited whole genome sequencing, with superior analytic performance for structural and copy number variants. An expert panel adjudicated the indications, contraindications, efficacy, and evidence-of-efficacy of 9911 drug, device, dietary, and surgical interventions for 563 severe, childhood, genetic diseases. The 421 (75%) diseases and 1527 (15%) effective interventions retained are integrated with 13 genetic disease information resources and appended to diagnostic reports ( https://gtrx.radygenomiclab.com ). This system provided correct diagnoses in four retrospectively and two prospectively tested infants. The Genome-to-Treatment system facilitates optimal outcomes in children with rapidly progressive genetic diseases.

First-in-class trispecific VHH-Fc based antibody with potent prophylactic and therapeutic efficacy against SARS-CoV-2 and variants.

SARS-CoV-2 and its variants have persisted in this ongoing COVID-19 pandemic. While the vaccines have greatly reduced the COVID-19 cases, hospitalizations, and death, about half of the world remain unvaccinated due to various reasons. Furthermore, the duration of the immunity gained from COVID-19 vaccination is still unclear. Therefore, there is a need for innovative prophylactic and treatment measures. In response to this need, we previously reported on the successful computer-aided development of potent VHH-based multispecific antibodies that were characterized in vitro. Here, we evaluated in vivo efficacy and safety of the lead trispecific VHH-Fc, ABS-VIR-001. Importantly, our data showed that ABS-VIR-001 treatment prevented SARS-CoV-2 infection and death when provided as an intranasal prophylaxis in a humanized ACE-2 mouse model. In addition, ABS-VIR-001 post-exposure treatment was shown to greatly reduce viral loads by as much as 50-fold. A detailed panel of metabolic and cellular parameters demonstrated that ABS-VIR-001 treatment was overall comparable to the PBS treatment, indicating a favorable safety profile. Notably, our inhibition studies show that ABS-VIR-001 continued to demonstrate unwavering efficacy against SARS-CoV-2 mutants, associated with key variants including Delta and Omicron, owing to its multiple epitope design. Lastly, we rigorously tested and confirmed the excellent thermostability of ABS-VIR-001 when heated to 45 °C for up to 4 weeks. Taken together, our study suggests that ABS-VIR-001 is an efficacious and durable prophylaxis and post-exposure treatment for COVID-19 with promising safety and manufacturability features for global distribution.

Uncertain, Not Unimportant: Callosal Dysgenesis and Variants of Uncertain Significance in ROBO1.

Congenital anomalies affect 3% to 5% of births and remain the leading cause of infant death in the United States. As whole exome and genome sequencing are increasingly used to diagnose underlying genetic disease, the patient's clinical presentation remains the most important context for interpreting sequencing results, including frequently reported variants of uncertain significance (VUS). Classification of a variant as VUS acknowledges limits on evidence to establish whether a variant can be classified as pathogenic or benign according to the American College of Medical Genetics guidelines. Importantly, the VUS designation reflects limits on the breadth of evidence linking the genetic variant to a disease. However, available evidence, although limited, may be surprisingly relevant in an individual patient's case. Accordingly, a VUS result should be approached neither as nondiagnostic genetic result nor as automatically "uncertain" in its potential to guide clinical decision-making. In this article, we discuss a case of an infant born at 29 weeks 4 days without a corpus callosum, whose whole genome sequencing yielded compound heterozygous variants both classified as VUS in ROBO1, a gene encoding for a receptor involved in a canonical signaling mechanism that guides axons across midline. Approaching the VUS result as potentially pathogenic, we found the infant ultimately had pituitary dysfunction and renal anomalies consistent with other reported ROBO1 variants and basic science literature. Accordingly, we highlight resources for variant interpretation available to clinicians to evaluate VUS results, particularly as they inform the diagnosis of individually rare but collectively common rare diseases.

Rare deleterious mutations of HNRNP genes result in shared neurodevelopmental disorders.

BACKGROUND: With the increasing number of genomic sequencing studies, hundreds of genes have been implicated in neurodevelopmental disorders (NDDs). The rate of gene discovery far outpaces our understanding of genotype-phenotype correlations, with clinical characterization remaining a bottleneck for understanding NDDs. Most disease-associated Mendelian genes are members of gene families, and we hypothesize that those with related molecular function share clinical presentations. METHODS: We tested our hypothesis by considering gene families that have multiple members with an enrichment of de novo variants among NDDs, as determined by previous meta-analyses. One of these gene families is the heterogeneous nuclear ribonucleoproteins (hnRNPs), which has 33 members, five of which have been recently identified as NDD genes (HNRNPK, HNRNPU, HNRNPH1, HNRNPH2, and HNRNPR) and two of which have significant enrichment in our previous meta-analysis of probands with NDDs (HNRNPU and SYNCRIP). Utilizing protein homology, mutation analyses, gene expression analyses, and phenotypic characterization, we provide evidence for variation in 12 HNRNP genes as candidates for NDDs. Seven are potentially novel while the remaining genes in the family likely do not significantly contribute to NDD risk. RESULTS: We report 119 new NDD cases (64 de novo variants) through sequencing and international collaborations and combined with published clinical case reports. We consider 235 cases with gene-disruptive single-nucleotide variants or indels and 15 cases with small copy number variants. Three hnRNP-encoding genes reach nominal or exome-wide significance for de novo variant enrichment, while nine are candidates for pathogenic mutations. Comparison of HNRNP gene expression shows a pattern consistent with a role in cerebral cortical development with enriched expression among radial glial progenitors. Clinical assessment of probands (n = 188-221) expands the phenotypes associated with HNRNP rare variants, and phenotypes associated with variation in the HNRNP genes distinguishes them as a subgroup of NDDs. CONCLUSIONS: Overall, our novel approach of exploiting gene families in NDDs identifies new HNRNP-related disorders, expands the phenotypes of known HNRNP-related disorders, strongly implicates disruption of the hnRNPs as a whole in NDDs, and supports that NDD subtypes likely have shared molecular pathogenesis. To date, this is the first study to identify novel genetic disorders based on the presence of disorders in related genes. We also perform the first phenotypic analyses focusing on related genes. Finally, we show that radial glial expression of these genes is likely critical during neurodevelopment. This is important for diagnostics, as well as developing strategies to best study these genes for the development of therapeutics.

Abnormal SCID Newborn Screening and Spontaneous Recovery Associated with a Novel Haploinsufficiency IKZF1 Mutation.

PURPOSE: IKAROS, encoded by IKZF1, is a member of the IKAROS family of zinc-finger transcription factors playing critical roles in lymphocyte development, differentiation, and tumor suppression. Several studies demonstrated that IKZF1 mutations affecting DNA binding or homo-/hetero-dimerization are mostly associated with common variable immunodeficiency, combined immunodeficiency, or hematologic manifestations. Herein we report a likely de novo, nonsense IKZF1 mutation (p.C182*) in a baby with low T cell receptor excision circles (TREC) identified by newborn screening testing for severe combined immunodeficiency. The patient also presented a profound B cell deficiency at birth. METHODS: Genetic, functional, immunologic, and clinical outcome data associated with this patient and her mutation were evaluated. RESULTS: Mutant p.C182* was detected in the cytoplasm of the patient's primary cells, in contrast to wild type (WT) IKAROS protein, only detected in the nucleus. Functional in vitro assessments revealed that p.C182* was less stable than WT IKAROS protein and failed to bind to its target DNA binding sequence and dimerize with WT IKAROS protein, resulting in impaired pericentromeric targeting and transcriptional repression by means of haploinsufficiency. During follow-up, while a spontaneous recovery of TREC and T cells was observed, B cells improved but not to sustained normal ranges. CONCLUSIONS: Patients with IKAROS-associated diseases can present with SCID-like TREC values through newborn screening testing. IKZF1 mutations should be added to the low TREC differential, although spontaneous recovery has to be considered.

Capsule carbohydrate structure determines virulence in Acinetobacter baumannii.

Acinetobacter baumannii is a highly antibiotic-resistant bacterial pathogen for which novel therapeutic approaches are needed. Unfortunately, the drivers of virulence in A. baumannii remain uncertain. By comparing genomes among a panel of A. baumannii strains we identified a specific gene variation in the capsule locus that correlated with altered virulence. While less virulent strains possessed the intact gene gtr6, a hypervirulent clinical isolate contained a spontaneous transposon insertion in the same gene, resulting in the loss of a branchpoint in capsular carbohydrate structure. By constructing isogenic gtr6 mutants, we confirmed that gtr6-disrupted strains were protected from phagocytosis in vitro and displayed higher bacterial burden and lethality in vivo. Gtr6+ strains were phagocytized more readily and caused lower bacterial burden and no clinical illness in vivo. We found that the CR3 receptor mediated phagocytosis of gtr6+, but not gtr6-, strains in a complement-dependent manner. Furthermore, hypovirulent gtr6+ strains demonstrated increased virulence in vivo when CR3 function was abrogated. In summary, loss-of-function in a single capsule assembly gene dramatically altered virulence by inhibiting complement deposition and recognition by phagocytes across multiple A. baumannii strains. Thus, capsular structure can determine virulence among A. baumannii strains by altering bacterial interactions with host complement-mediated opsonophagocytosis.

Characterization of Pneumococcal Colonization Dynamics and Antimicrobial Resistance Using Shotgun Metagenomic Sequencing in Intensively Sampled South African Infants.

Background: There remains a significant proportion of deaths due to pneumococcal pneumonia in infants from low- and middle-income countries despite the marginal global declines recorded in the past decade. Monitoring changes in pneumococcal carriage is key to understanding vaccination-induced shifts in the ecology of carriage, patterns of antimicrobial resistance, and impact on health. We longitudinally investigated pneumococcal carriage dynamics in PCV-13 vaccinated infants by collecting nasopharyngeal (NP) samples at 2-weekly intervals from birth through the first year of life from 137 infants. As a proof of concept, 196 NP samples were retrieved from a subset of 23 infants to explore strain-level pneumococcal colonization patterns and associated antimicrobial-resistance determinants. These were selected on the basis of changes in serotype and antibiogram over time. NP samples underwent short-term enrichment for streptococci prior to total nucleic acid extraction and whole metagenome shotgun sequencing (WMGS). Reads were assembled and aligned to pneumococcal reference genomes for the extraction of pneumococcal and non-pneumococcal bacterial reads. Pneumococcal contigs were aligned to the Antibiotic Resistance Gene-ANNOTation database of acquired AMR genes. In silico pneumococcal capsular and multilocus sequence typing were performed. Results: Of the 196 samples sequenced, 174 had corresponding positive cultures for pneumococci, of which, 152 were assigned an in silico serotype. Metagenomic sequencing detected a single pneumococcal serotype in 85% (129/152), and co-colonization in 15% (23/152) of the samples. Twenty-two different pneumococcal serotypes were identified, with 15B/15C and 16F being the most common non-PCV13 serotypes, while 23F and 19A were the most common PCV13 serotypes. Twenty-six different sequence types (STs), including four novel STs were identified in silico. Mutations in the folA and folP genes, associated with cotrimoxazole resistance, were detected in 89% (87/98) of cotrimoxazole-non-susceptible pneumococci, as well as in the pbp1a and pbp2x genes, in penicillin non-susceptible ST705215B/15C isolates. Conclusions: Metagenomic sequencing of NP samples is a valuable culture-independent technique for a detailed evaluation of the pneumococcal component and resistome of the NP microbiome. This method allowed for the detection of novel STs, as well as co-colonization, with a predominance of non-PCV13 serotypes in this cohort. Forty-eight resistance genes, as well as mutations associated with resistance were detected, but the correlation with phenotypic non-susceptibility was lower than expected.

Development of humanized tri-specific nanobodies with potent neutralization for SARS-CoV-2.

SARS-CoV-2 is a newly emergent coronavirus, which has adversely impacted human health and has led to the COVID-19 pandemic. There is an unmet need to develop therapies against SARS-CoV-2 due to its severity and lack of treatment options. A promising approach to combat COVID-19 is through the neutralization of SARS-CoV-2 by therapeutic antibodies. Previously, we described a strategy to rapidly identify and generate llama nanobodies (VHH) from naïve and synthetic humanized VHH phage libraries that specifically bind the S1 SARS-CoV-2 spike protein, and block the interaction with the human ACE2 receptor. In this study we used computer-aided design to construct multi-specific VHH antibodies fused to human IgG1 Fc domains based on the epitope predictions for leading VHHs. The resulting tri-specific VHH-Fc antibodies show more potent S1 binding, S1/ACE2 blocking, and SARS-CoV-2 pseudovirus neutralization than the bi-specific VHH-Fcs or combination of individual monoclonal VHH-Fcs. Furthermore, protein stability analysis of the VHH-Fcs shows favorable developability features, which enable them to be quickly and successfully developed into therapeutics against COVID-19.