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Background: Deciphering hypoplastic left heart syndrome (HLHS) pathogenesis is confounded by its genetic heterogeneity and oligogenic underpinnings. Methods: Whole genome sequences were analyzed by 3 independent strategies to identify HLHS gene candidates, ranked by variant, gene, and disease-level metrics. Results: First, a genome-wide association study of 174 cases and 853 controls revealed suggestive association with a MYO18B intron 33 variant (rs2269628-G; frequency=0.55 versus 0.39; OR, 1.97 [95% CI, 1.54–2.52]; P =6.70×10 − 8 ). Second, transmission disequilibrium testing of 161 HLHS proband-parent trios revealed overrepresentation of a MYO18B intron 42 variant (rs73154186-A; frequency=0.05; OR, 24 [95% CI, 3.2–177.4]; P =4.23×10 − 6 ). Third, rare, predicted-damaging variants were filtered in 2 multiplex families. In 141H, 2 fifth-degree relatives with HLHS shared a paternally-inherited MYO5A missense variant (p.Arg801Trp; frequency=0.00003; combined annotation-dependent depletion score=29), each with a maternally-inherited or de novo candidate modifier variant in a MYO5A-interacting conventional myosin. In 442H, a HLHS proband was compound heterozygous for MYO15A variants—a maternally-inherited pathogenic stop-gain variant co-segregating with tetralogy of Fallot and bicuspid aortic valve in maternal relatives (p.Tyr2819Ter; frequency=0.00003) and a paternally-inherited intronic variant altering a canonical transcription factor binding site (rs1277068603; frequency=0.00001; position weight matrix score=0.98). Conclusions: Collectively, these findings suggest that common and rare alleles within unconventional myosin genes are associated with HLHS susceptibility. The identified candidate MYO18B regulates cardiac sarcomerogenesis, supporting the hypothesis of intrinsic myogenic perturbation in arrested left heart development.

Purpose: Planning target volume (PTV) expansion for post-prostatectomy radiotherapy is typically ≥5 mm. Recent clinical trials have proved the feasibility of a reduced margin of 2–3 mm for treatments on MRI-linac. We aim to study the minimum PTV margin needed using iterative cone-beam CT (iCBCT) as image guidance on conventional linacs. Materials/Methods: Fourteen patients who received post-prostatectomy irradiation (8 with an endorectal balloon and 6 without a balloon) were included in this study. Treatment was delivered with volumetric modulated radiation therapy (VMAT). Fractional dose delivery was evaluated in 165 treatment fractions. The bladder, rectal wall, femoral heads, and prostate bed clinical tumor volume (CTV) were contoured and verified on daily iCBCT. PTV margins (0 mm, 2 mm, and 4 mm) were evaluated on daily iCBCT. CTV coverage and OAR dose parameters were assessed with each PTV margin. Results: CTV D100% was underdosed with a 0 mm margin in 32% of fractions in comparison with 2 mm (6%) and 4 mm (6%) PTV margin (p ≤ 0.001). CTV D95% > 95% was met in 93–94% fractions for all PTV expansions. CTV D95% > 95% was achieved in more patients with an endorectal balloon than those without: 0 mm—90/91 (99%) vs. 63/74 (85%); 2 mm—90/91 (99%) vs. 65/75 (87%); 4 mm—90/90 (100%) vs. 63/73 (86%). There was no difference in absolute median change in CTV D95% (0.32%) for 0-, 2-, and 4 mm margins. The maximum dose remained under 108% for 100% (0 mm), 97% (2 mm), and 98% (4 mm) of images. Rectal wall maximum dose remained under 108% for 100% (0 mm), 100% (2 mm), and 98% (4 mm) of images. Conclusions: With high-quality iCBCT image guidance, PTV margin accounting for inter-fractional uncertainties can be safely reduced for post-prostatectomy radiotherapy. For fractionated radiotherapy, an isotropic expansion of 2 mm and 4 mm may be considered for margin expansion with and without the endorectal balloon. Future application for margin reduction needs to be further evaluated and considered with the advent of shorter post-prostatectomy radiation courses.

Abstract Obesity is associated with several skeletal muscle impairments which can be improved through an aerobic exercise prescription. The possibility that exercise responsiveness is diminished in people with obesity has been suggested but not well‐studied. The purpose of this study was to investigate how obesity influences acute exercise responsiveness in skeletal muscle and circulating amino metabolites. Non‐obese (NO; n = 19; 10F/9M; BMI = 25.1 ± 2.8 kg/m 2 ) and Obese (O; n = 21; 14F/7M; BMI = 37.3 ± 4.6 kg/m 2 ) adults performed 30 min of single‐leg cycling at 70% of VO 2 peak. 13 C 6 ‐Phenylalanine was administered intravenously for muscle protein synthesis measurements. Serial muscle biopsies (vastus lateralis) were collected before exercise and 3.5‐ and 6.5‐h post‐exercise to measure protein synthesis and gene expression. Targeted plasma metabolomics was used to quantitate amino metabolites before and 30 and 90 min after exercise. The exercise‐induced fold change in mixed muscle protein synthesis trended ( p = 0.058) higher in NO (1.28 ± 0.54‐fold) compared to O (0.95 ± 0.42‐fold) and was inversely related to BMI (R 2 = 0.140, p = 0.027). RNA sequencing revealed 331 and 280 genes that were differentially expressed after exercise in NO and O, respectively. Gene set enrichment analysis showed O had six blunted pathways related to metabolism, cell to cell communication, and protein turnover after exercise. The circulating amine response further highlighted dysregulations related to protein synthesis and metabolism in adults with obesity at the basal state and in response to the exercise bout. Collectively, these data highlight several unique pathways in individuals with obesity that resulted in a modestly blunted exercise response.

AbstractBackgroundAlzheimer’s disease (AD) is a progressive neurodegenerative disorder neuropathologically characterized by amyloid‐β (Aβ) plaques and tau neurofibrillary tangles often measured by Thal phase and Braak stage, respectively. Frequently, Aβ also deposits in the brain vasculature with severity categorized by a cerebral amyloid angiopathy (CAA) score. Assessment of these and related endophenotypes can reveal biological insights into the phenotypic variability in AD. We hypothesize that, amongst AD patients, severity of neuropathology and brain levels of AD‐related proteins are influenced by variation in DNA methylation (DNAm), the identification of which may uncover biological pathways for AD pathogenesis. To test this, we performed epigenome‐wide association studies (EWAS) using DNAm from the temporal cortex (TCX) and cerebellum (CER) with AD‐related neuropathology measures (Braak, Thal, and CAA) and brain levels of five proteins (apoE, Aβ40, Aβ42, tau, and p‐tau).MethodsDNAm from autopsy‐confirmed AD cases available through the Mayo Clinic Brain Bank was measured by reduced representation bisulfite sequencing (RRBS) from 471 brain samples, 200 of which had both TCX and CER measures. Bisulfite converted reads were sequenced on the Illumina High Seq4000 and quality filtered. Neuropathology scoring was performed by an experienced neuropathologist. TCX levels of five AD‐related proteins from three fractions (buffer‐soluble, detergent‐soluble, and insoluble) were measured by ELISA (Liu et al. 2020). Individual CpG methylation ratios were tested for association with each neuropathological and biochemical measure using the R package CpGassoc.ResultsWe identified multiple epigenome‐wide significant CpGs unique to each AD‐related measure. Comparisons between the TCX and CER revealed region specific CpG associations. The effects of sex, age at death, APOE genotype, and presence of genetic variants for each significant CpG association were also identified.ConclusionsAlthough all neuropathological and biochemical measures tested reflect core AD pathologies, our results demonstrate unique associations of epigenetic factors with these individual phenotypes, suggesting that their variability may be governed by distinct epigenetic processes. Discovery of DNAm changes that underlie specific neuropathological or biochemical outcomes can enhance our understanding of AD pathophysiology and may lead to the identification of novel gene or pathway targets for the different facets of this complex condition.

AbstractBackgroundInter‐cellular communication within the gliovascular unit (GVU) is critical for cerebral blood flow regulation, maintenance of the blood‐brain‐barrier (BBB) properties, and brain energy metabolism. Although numerous cell biology studies demonstrate the role of the GVU for BBB breakdown in Alzheimer’s disease (AD), the precise molecular changes contributing to its pathophysiology are unclear.MethodWe performed single nucleus RNA sequencing (snRNAseq) of temporal cortex tissue in 24 AD and control brains to understand the transcriptional changes in the cells of GVU. We acquired the snRNAseq profile of 79,751 total brain cells that comprise 6,541 astrocytes and 2,210 vascular cells. The latter formed three distinct vascular clusters characterized as activated pericytes, endothelia and resting pericytes. We identified differentially expressed genes (DEGs) and their enriched pathways in these clusters and detected the most transcriptional changes within activated pericytes. Using our data and a knowledge‐based predictive algorithm, we discovered and prioritized molecular interactions between vascular and astrocyte clusters, the main cell types of the GVU of the BBB.ResultsVascular targets predicted to interact with astrocytic ligands have biological functions in signalling, angiogenesis, amyloid ß metabolism and cytoskeletal structure. Top astrocytic and vascular interacting molecules include both novel and known AD risk genes such as APOE, APP and ECE1. We validated gene expression changes using qPCR. We are in the process of evaluating these gene expression changes in antemortem blood from AD and control participants.ConclusionOur findings provide valuable information into the transcriptional changes in predicted vascular‐astrocytic partners at the GVU, highlighting insights into the molecular mechanisms of BBB breakdown in AD.

Abstract The COVID‐19 pandemic and its accompanying infection control measures introduced sudden and significant disruptions to the lives of children and adolescents around the world. Given the potential for negative impacts on the mental health of youths as a result of these changes, we conducted a systematic review and meta‐analysis to examine the prevalence of depressive symptoms, anxiety symptoms, and sleep disturbances in children and adolescents during the pandemic. We searched major literature databases for relevant cross‐sectional or longitudinal studies that included primary and secondary school students or children and adolescents ≤18 years of age. Prevalence values were extracted, logit‐transformed, and pooled. Based on 191 included studies with 1,389,447 children and adolescents, we found the pooled prevalence of depressive symptoms, anxiety symptoms, and sleep disturbances to be 31%, 31%, and 42%, respectively. Age, grade levels, education levels, gender, geographical regions, and electronics use were correlated with the prevalence of mental health symptoms. The prevalence of mental health symptoms also increased with time, although signs of recovery and stabilization were also observed. Overall, the results from this review demonstrate the need for increased mental health research, monitoring, and intervention for children and adolescents during the current and future pandemics.

Abstract Background Pneumocystis jirovecii pneumonia (PCP) is a potentially deadly infection afflicting the immunocompromised population, including solid organ transplant recipients. Several risk factors have been described, including acute rejection, lymphopenia, and cytomegalovirus (CMV) infection. However, little is known regarding the risk imparted by posttransplant lymphoproliferative disorder (PTLD). Methods We performed a nested case-control study of solid organ transplant recipients diagnosed with PCP from 2000-2020. PCP was defined as positive smear or polymerase chain reaction testing with compatible clinical symptoms and radiographic findings. Two control were matched to each case by year of first transplant, first transplanted organ, and sex. Each control had at least as much follow-up from their transplant date to their matched case’s PCP diagnosis date. Multivariable conditional logistic regression was performed to analyze theorized risk factors. Results Sixty-seven cases met inclusion criteria and were matched to 134 controls (Table 1). Median age was 60.9 years, and the most common transplant type was kidney (52.2%). Fourteen patients had a history of PTLD, 12 of which developed PCP. All cases of PTLD were monomorphic, 6 were EBV-positive, 9 were receiving chemotherapy at the index date, and only 1 control patient was receiving PCP prophylaxis. The cases with PTLD developed PCP a median of 85 days after PTLD diagnosis, while the two controls were diagnosed more than 1 year earlier. After adjusting for age, acute rejection requiring treatment within the last 6 months, CMV infection within 6 months, current PCP prophylaxis, and lymphopenia (lymphocyte count < 0.5 x109/L) within 6 months, PTLD had a significant association with PCP (OR 14.0, 95% CI 1.7-114.5; p = .014). Lymphopenia was also associated with PCP (OR 8.2, 95% CI 3.2-20.7; p < .001), while the other factors were not. Table 1:Characteristics of 67 Solid Organ Transplant Recipients with Pneumocystis Pneumonia and 134 Matched Controls Conclusion Diagnosis of PTLD is independently associated with subsequent PCP after adjustment for recognized risk factors. This association is likely influenced by PTLD-directed chemotherapy, particularly regimens containing rituximab. PCP prophylaxis should be initiated in solid organ transplant recipients with PTLD, particularly those undergoing active therapy, and those with severe lymphopenia. Disclosures All Authors: No reported disclosures.

Abstract Background Posaconazole therapeutic drug monitoring (TDM) is widely utilized to assess therapeutic efficacy and safety (i.e., hepatotoxicity, QTc prolongation); however, little is known about clinical effects of very high serum posaconazole serum levels (i.e., ≥ 5000 mg/mL). Reported incidence rate of adverse drug reactions (ADRs) with posaconazole per most recent clinical trial was ∼30%. The primary objective was to compare the ADR incidence in patients with serum posaconazole levels of 3000-4999 ng/mL to ≥5000 ng/mL. Methods This retrospective cohort study included adult patients with a posaconazole serum level ≥3000 ng/mL from 1/1/2019 to 04/30/2021. The primary outcome was symptomatic ADR at time of first serum level ≥3000 ng/mL. Secondary outcomes were laboratory defined hepatoxicity, electrolyte and adrenal laboratory abnormalities, QTc changes, and dose changes in response to TDM. Patient outcomes were censored after the first serum level and were compared between groups using Fisher’s exact tests. Results Ninety patients met inclusion criteria, 80 with a level of 3000-4999 ng/mL and 10 with a level of ≥ 5000 ng/mL occurring at a median of 91 days (26-443) and 27 days (12-45) from posaconazole initiation, respectively. Majority of patients were immunocompromised (55.6% transplant recipients, 28.9% active malignancy, 5.6% other) with a split of treatment (50%) and prophylaxis (42.2%) indication. Symptomatic ADRs were very common in patients with posaconazole levels of ≥5000 ng/mL and 3000-4999 ng/mL (80% vs. 58.8%; p=0.31), primarily neurologic (49.1% overall) followed by gastrointestinal (32.7% overall). Hepatotoxicity was also common (≥5000 ng/mL 40% vs 3000-4999 ng/mL 23.4%, p=0.26). Fifty percent of patients had the posaconazole dose continued without change. Electrolytes and QTc results were similar between groups, but median overall QTc was borderline high (456 [IQR 435, 479]). Conclusion There are safety concerns for patients with serum posaconazole levels ≥3000 ng/mL. Posaconazole levels should be monitored and, importantly, dose adjusted according to serum level and patient symptoms for both treatment and prophylaxis indications. Disclosures Christina G. Rivera, PharmD, Gilead: Grant/Research Support|Gilead: Honoraria|Insmed: Honoraria.