PRDM16 Regulates Leukemic Stemness, Metabolism, and Quiescence in NUP98::NSD1/WT1ko AML Part 1

BACKGROUND:Agents such as digoxin and potassium chloride are frequently administered to arrest fetal circulation prior to termination procedures. Although this practice is common, the impact of delayed evacuation on human fetal tissue quality has not been systematically assessed. High-quality tissue is essential for accurate diagnosis of fetal abnormalities and for biomedical research. OBJECTIVE:To evaluate the effects of induced fetal demise and delayed evacuation on human fetal tissue quality across multiple organ systems, and to determine whether digoxin independently contributes to tissue degradation. STUDY DESIGN:Tissue was collected from second-trimester dilation and evacuation procedures performed either after induced demise with digoxin or potassium chloride followed by evacuation 19-25 hours later, or without prior induction (immediate evacuation). Brain, lung, kidney, liver, and muscle were evaluated for morphology, proliferation, apoptosis, cell viability, culture success, and nucleic acid integrity. To model in utero conditions, tissues were incubated at 37 °C or 4 °C for up to 18 hours, with and without digoxin. RESULTS:Delayed evacuation was associated with structural disruption, reduced proliferation, increased apoptosis, diminished fibroblast viability, and reduced RNA integrity compared to immediate evacuation. DNA integrity remained robust, with all samples suitable for PCR. Digoxin alone did not significantly alter RNA quality; however, when combined with prolonged incubation at 37 °C, RIN and DV200 values were significantly reduced across multiple tissues. CONCLUSION:Warm ischemia following circulatory arrest is the principal driver of tissue degradation, with RNA and cell viability most affected and DNA relatively preserved. Digoxin alone has minimal effect but accelerates degradation under warm conditions. Minimizing the interval between circulatory arrest and evacuation, and avoiding prolonged exposure to body temperature, may preserve tissue integrity and improve the diagnostic and research utility of HFT.

Human fetal tissue (HFT) has played a pivotal role in many areas of biomedical research. Since its inception, HFT research has been a topic of ethical debate and specific regulation due to its link to abortion. To date, the ethical debate surrounding HFT donation for research has proceeded with limited empirical data to guide policy decisions. Current regulations are designed to minimize the possibility that HFT donation could impact the decision of whether to have an abortion by requiring that clinical consent for abortion be obtained prior to discussing research opportunities. Despite these timing restrictions, questions continue to be raised about the validity of informed consent for HFT research. We sought to collect empirical data to inform the policy debates by directly querying the experience of pregnant women asked to donate HFT for research. We conducted qualitative interviews with 51 women who chose (n = 40), or declined (n = 11), to donate HFT. Recruitment challenges and high attrition impacted the number of participants we were able to interview who had declined to donate. Results revealed that none of the interviewees felt pressured to donate, and no one objected to being asked to participate. The majority of those who chose to donate HFT felt invested in the research and motivated to learn results. Participant feedback about the consent process, however, converged on a desire to learn about the option of HFT donation earlier in the appointment to allow for more time to consider research participation. Limitations include that the study took place in a single US state and results may differ in other jurisdictions. We conclude with recommendations for policy amendments that could improve the experience of potential participants asked to donate HFT for biomedical research.

PURPOSE:Protein-altering gene variants in SEPSECS disrupt the biosynthesis of selenoproteins, leading to a spectrum of neurological diseases. METHODS:We studied 27 individuals with biallelic SEPSECS variants, identifying 13 unreported gene variants. To better understand and diagnose the disorder, broad biochemical correlation of neurological symptoms, focused metabolomics, and structural and in vitro activity analyses were deployed. RESULTS:Our results suggest three general clinical courses: (i) severe early-onset with cerebellar or cerebral atrophy, (ii) milder early-onset with gradual deterioration, and (iii) late-onset, mild disease. SEPSECS variants primarily affect the brain. In only one individual out of eight, thyroid hormone measurements suggested a defect of T4 to T3 conversion. Accompanied increase in glutathione and sulfur metabolites in plasma indicates elevated oxidative stress. Variants mapping to conserved N- and C-termini and catalytic site elicit SEPSECS misfolding, aggregation, thermal instability, and loss of function, that could ultimately lead to ferroptosis of neurons and perhaps oligodendrocytes. For differential diagnosis and monitoring therapeutic attempts, we recommend measuring levels of plasma selenium, glutathione and sulfur metabolites, GPX activity, and SELENOP. Given the pontine involvement in less than half of the cases, we suggest renaming the syndrome from PCH2D to SEPSECS-related neurodevelopmental disorder. CONCLUSION:Our study expands the understanding of SEPSECS-related neurodevelopmental disorders, highlighting the need for updated diagnostic criteria and potential treatment strategies.

Fetal-Oncogenic Gene Signature in NUP98::NSD1/WT1ko AML

Venetoclax Targets OXPHOS and BCL2 Dependency in Fetal-Origin NUP98::NSD1/WT1ko AML

Single-Cell Analysis Reveals Primitive Stem and Progenitor Expansion in NUP98::NSD1 Leukemia

Schematic Model of Leukemia Initiation and Therapy Response in NUP98::NSD1 AML Based on Developmental Origin

Primary cilia are small organelles acting as cellular antennae that sense and transduce diverse signals, including developmental signaling pathways in the developing central nervous system (CNS). This signaling is essential for normal CNS development, as evidenced by the prevalence of neurodevelopmental phenotypes in conditions arising from primary cilia dysfunction (ciliopathies). Though significant research has focused on the roles of primary cilia during CNS development, the functions of primary cilia in the mature CNS have only recently become a focus of investigation. Primary cilia are present on most vertebrate cells, including mature neurons and astrocytes and reportedly localize G-protein-coupled receptors, voltage-gated ion channels, and even synaptic proteins. Moreover, recent evidence highlights the dense "contactome" of cilia in the brain with adjacent neuronal structures and has even identified axo-ciliary synapses. Primary cilia are therefore both perfectly equipped and positioned to participate in regulating mature neural circuits. Consistent with these observations, primary cilia have also been linked to neurological and psychiatric symptoms without underlying brain malformations, both in ciliopathies and in nonciliopathy neurological conditions like autism spectrum disorder and schizophrenia. In this review, we bridge insights from human disease to evidence gained from animal and cell models to highlight the evolving roles of primary cilia in the developing and mature CNS. Primary cilia in the developing brain act as classical cellular antennae sensing secreted ligands, while primary cilia in the mature brain may also be capable of contact-dependent signaling, indicating a potential shift in the signaling capacity of primary cilia in the CNS.

WT1 Loss Amplifies Stemness, EGR1 and MYC Expression in NUP98::NSD1 AML

INTRODUCTION:As genomic sequencing (GS) is integrated into clinical medicine, understanding how patients and families perceive its utility is critical. Conceptual models for perceived utility have been developed, however, direct application of such models to independently-collected qualitative data remains rare. To fill this gap, we translated a comprehensive model of perceived utility (Smith et al. 2022) into a codebook and revised it via application to a qualitative dataset capturing parent-reported utility of GS. METHODS:Using the domains and subdomains from Smith et al.'s conceptual model, we developed an initial codebook. We tested the derived codebook on data from 40 interviews with parents of children enrolled in SeqFirst, a research study offering GS for critically ill newborns in the neonatal intensive care unit and young children (<3 years) with unexplained developmental differences. Four coders independently applied the codebook. We addressed ambiguities through memoing and refined definitions via study team consensus. RESULTS:The resulting codebook includes all five of Smith et al.'s (2022) domains: clinical, emotional, behavioral, cognitive, and social utility. We renamed and relocated several subdomains to add additional clarity in coding decisions, and developed definitions for all subdomain codes. The final codebook is included as a resource for future research. CONCLUSIONS:This study translates a conceptual framework of perceived utility of GS into a practical codebook for qualitative research. Our intent is to facilitate conceptually-grounded, replicable coding across studies and settings, supporting both inductive and deductive analyses. Applying this codebook can enhance comparability of qualitative findings, inform refinement of conceptual models, and guide the further development of patient-centered measures of GS utility.

Transcriptional Profiling upon Treatment in NUP98::NSD1 AML

Background:The absence of standardized approaches for handling genetic test results in electronic health records (EHRs), combined with a lack of diagnostic codes for most rare disorders, hinders accurate and timely identification of patients with rare genetic variants. This impedes access to research opportunities and genomic-driven care. To reduce the diagnostic odyssey, identify research-eligible subjects, and ultimately enhance patient care, it is critical to optimize approaches to retrieve genetic results. Objectives:To characterize resource requirements, yield, and biases among methods for identifying and retrieving genetic test results across 11 Intellectual and Developmental Disability Research Centers (IDDRC). Design:A survey was used to collect details from the authors on approaches to identify EHRs from patients who had genetic testing and variants of interest were reported; surveys were completed in 2022. Methods:Strengths and limitations in approaches to identify and retrieve genetic test results conducted from the implementation of EHR systems were evaluated. A standard template was used to collect genetic testing storage formats, methods to identify patients with rare disease variants, estimates of time/cost, nature of accessed data, method-specific bias in types of American College of Medical Genetics and Genomics classified variants identified. When possible, precision when performing gene name searches in the EHR was calculated. Results:Four approaches were used: (1) manual searches, reviews, and extractions, (2) natural language processing software-aided manual reviews and extractions, (3) custom databases via testing lab collaborations, and (4) testing EHR vendor-designed genomics modules. The fully manual approach required minimal infrastructure and allowed access to clinical notes but missed variants of unknown clinical significance. Precision for gene name matches based on searches of 59 genes was 0.16. Natural language processing software minimized effort but required considerable informatics support. Custom databases and EHR vendor modules necessitated substantial computational support; however, genetic testing results retrieval was efficient. Conclusion:Leveraging the IDDRC network, we found that methods to store, search and extract genetic testing results vary widely, especially regarding older test results, and have distinct benefits and limitations. Limitations are best addressed through practice guidelines that standardize storage and retrieval of genetic test results to facilitate efficient identification of research eligible subjects and genomic-informed patient care.

BACKGROUND:Decisions to split two or more phenotypic manifestations related to genetic variations within the same gene can be challenging, especially during the early stages of syndrome discovery. Genotype-based diagnostics with artificial intelligence (AI)-driven approaches using next-generation phenotyping (NGP) and DNA methylation (DNAm) can be utilized to expedite syndrome delineation within a single gene. METHODS:We utilized an expanded cohort of 56 patients (22 previously unpublished individuals) with truncating variants in the MN1 gene and attempted different methods to assess plausible strategies to objectively delineate phenotypic differences between the C-Terminal Truncation (CTT) and N-Terminal Truncation (NTT) groups. This involved transcriptomics analysis on available patient fibroblast samples and AI-assisted approaches, including a new statistical method of GestaltMatcher on facial photos and blood DNAm analysis using a support vector machine (SVM) model. FINDINGS:RNA-seq analysis was unable to show a significant difference in transcript expression despite our previous hypothesis that NTT variants would induce nonsense mediated decay. DNAm analysis on nine blood DNA samples revealed an episignature for the CTT group. In parallel, the new statistical method of GestaltMatcher objectively distinguished the CTT and NTT groups with a low requirement for cohort number. Validation of this approach was performed on syndromes with known DNAm signatures of SRCAP, SMARCA2 and ADNP to demonstrate the effectiveness of this approach. INTERPRETATION:We demonstrate the potential of using AI-based technologies to leverage genotype, phenotype and epigenetics data in facilitating splitting decisions in diagnosis of syndromes with minimal sample requirement. FUNDING:The specific funding of this article is provided in the acknowledgements section.

Whole-genome sequencing (WGS) as a diagnostic test offers children suspected of having a rare genetic condition and their families the best direct path toward securing a precise genetic diagnosis (PrGD). Yet, a limited supply and inequitable access to genetic services are impediments to realizing the benefits of a PrGD. Such access disparities might be due to a range of structural and social determinants that manifest in interactions, or the lack thereof, between families, providers, and institutions. Semi-structured key informant interviews (n = 19) were conducted with neonatologists and neurodevelopmental clinic providers (NDV providers) who referred families to the SeqFirst study to identify barriers and inform strategies to improve equitable access to a PrGD via WGS. Overall, neonatologists and NDV providers were enthusiastic about offering WGS to their patients and families despite different contexts of medical care. Providers cited several considerations that influenced their introduction of WGS and genetic testing to families, including their perceptions of families' capacity, readiness, and distrust and the establishment of sufficient provider-family rapport. These considerations influenced providers' timing and introduction of genetic testing and WGS to families. Together, these findings suggest that providers' perceptions of families may result in delayed introduction of WGS.‬ Despite enthusiasm for early WGS across medical subspecialties, providers' perceptions of families and their social contexts highlight both challenges and opportunities in the implementation of WGS to promote and maximize equitable access.

The prefrontal cortex (PFC) is critical for myriad high-cognitive functions and is associated with several neuropsychiatric disorders. Here, using Patch-seq and single-nucleus multiomic analyses, we identified genes and regulatory networks governing the maturation of distinct neuronal populations in the PFC of rhesus macaque. We discovered that specific electrophysiological properties exhibited distinct maturational kinetics and identified key genes underlying these properties. We unveiled that RAPGEF4 is important for the maturation of resting membrane potential and inward sodium current in both macaque and human. We demonstrated that knockdown of CHD8, a high-confidence autism risk gene, in human and macaque organotypic slices led to impaired maturation, via downregulation of key genes, including RAPGEF4. Restoring the expression of RAPGEF4 rescued the proper electrophysiological maturation of CHD8-deficient neurons. Our study revealed regulators of neuronal maturation during a critical period of PFC development in primates and implicated such regulators in molecular processes underlying autism.

AIM:To identify the behavioral correlates of caregiver-reported oral health of individuals with Joubert Syndrome (JS). METHODS:This cross-sectional study included 302 caregivers of individuals with JS from the JS registry who responded to a 56-item REDCap survey, and a purposive subset of 30 participants with JS for which a dental screening was completed. The primary outcome was caregiver-reported oral health of individuals with JS (good/very good vs. fair/poor). Logistic regression models were used to generate confounder-adjusted odds ratios (OR) and evaluate associations between oral health behaviors and caregiver-reported oral health of individuals with JS (p < 0.05). RESULTS:The mean age of individuals with JS was 14.6 years (SD: 10.3). Most caregivers were female (84.1%), White (87.4%), non-Hispanic (91.1%), and reported annual household incomes above $100 000 (53.7%). Behaviors significantly associated with poorer oral health included not eating or drinking by mouth (OR = 4.52, p = 0.005), not toothbrushing daily (OR = 5.29, p < 0.001), and infrequent toothpaste use (OR = 3.68, p = 0.003). Screenings showed 40.0% had poor hygiene, 23.3% had untreated caries, and 46.7% had gingivitis. CONCLUSIONS:Future efforts should focus on improving oral health behaviors to prevent dental disease in individuals with JS.

Microtubule-actin cross-linking factor 1 (MACF1) is a large protein of the spectraplakin family, which is essential for brain development. MACF1 interacts with microtubules through the growth arrest-specific 2 (Gas2)-related (GAR) domain. Heterozygous MACF1 missense variants affecting the zinc-binding residues in this domain result in a distinctive cortical and brain stem malformation. Evidence for other MACF1-associated disorders is still limited. Here, we present a cohort of 45 individuals with heterozygous or bi-allelic MACF1 variants to explore the phenotypic spectrum and assess possible pathogenic relevance. We observe that de novo heterozygous missense variants in the EF-hand domains also result in distinctive brain malformation and provide experimental evidence that variants in the EF-hand/GAR module increase microtubule binding, suggestive of a toxic gain of function. Notably, no phenotype-genotype correlation was possible for the remaining heterozygous variants in other domains. A clinical review of eight families with bi-allelic variants reveals a possible complex neurodevelopmental syndrome of the central and peripheral nervous systems. In these individuals, bi-allelic variants mostly affect the Plakin domain. Furthermore, RNA sequencing and chromatin immunoprecipitation (ChIP) analyses of human fetal brain tissue reveal five MACF1 isoforms with region-specific expression, differing in their exon 1 transcription start sites but splicing to a common exon 2. This differential expression explains the frontal-predominant lissencephaly in an individual with a homozygous stop-gain in exon 1 (MACF1-204: c.70C>T [p.Arg24∗]), as this isoform is preferentially expressed in the frontal cortex. We conclude that MACF1-related disorders are strictly linked to domain function and the level of transcript expression, explaining the observed wide clinical heterogeneity.