Normal Gene Research Articles

Reference genes for gene expression profiling in mouse models of Listeria monocytogenes infection.

RT-qPCR dissects transcription-based processes but requires reference genes (RGs) for data normalization. This study prospected RGs for mouse macrophages (pMØ) and spleen infected with Listeria monocytogenes. ThepMØ were infected in vitro with L. monocytogenes or vehicle for 4h. Mice were injected with L. monocytogenes (or vehicle) and euthanized 24h post-injection. TheRGs came from a multispecies primer set, from theliterature or designed here. The RG ranking relied on GeNorm, NormFinder, BestKeeper, Delta-CTand RefFinder. B2m-H3f3a-Ppia were the most stable RGs for pMØ, albeit RG indexes fine-tuned estimations of cytokine relative expression. Actβ-Ubc-Ppia were the best RGs for spleen butmodestly impacted the cytokine relative expression. Hence, mouse models of L. monocytogenes require context-specific RGs for RT-qPCR, thus reinforcing its paramount contribution to accurate gene expression profiling.

Regulating PCCA gene expression by modulation of pseudoexon splicing patterns to rescue enzyme activity in propionic acidemia

Pseudoexons are nonfunctional intronic sequences that can be activated by deep-intronic sequence variation. Activation increases pseudoexon inclusion in mRNA and interferes with normal gene expression. The PCCA c.1285-1416A>G variation activates a pseudoexon and causes the severe metabolic disorder propionic acidemia by deficiency of the propionyl-CoA carboxylase enzyme encoded by PCCA and PCCB. We characterized this pathogenic pseudoexon activation event in detail and identified hnRNP A1 to be important for normal repression. The PCCA c.1285-1416A>G variation disrupts an hnRNP A1-binding splicing silencer and simultaneously creates a splicing enhancer. We demonstrate that blocking this region of regulation with splice-switching antisense oligonucleotides restores normal splicing and rescues enzyme activity in patient fibroblasts and in a cellular model created by CRISPR gene editing. Interestingly, the PCCA pseudoexon offers an unexploited potential to upregulate gene expression because healthy tissues show relatively high inclusion levels. By blocking inclusion of the nonactivated wild-type pseudoexon, we can increase both PCCA and PCCB protein levels, which increases the activity of the heterododecameric enzyme. Surprisingly, we can increase enzyme activity from residual levels in not only patient fibroblasts harboring PCCA missense variants but also those harboring PCCB missense variants. This is a potential treatment strategy for propionic acidemia.

Protamine 2 and phospholipase C zeta 1 are possible biomarkers for the diagnosis of male subfertility in frozen-thawed stallion semen

Subfertility is one of the main issues in horse breeding and the study of mRNAs in sperm might help in elucidating the reasons that lead to this diagnosis. The present study aims at assessing the differences in the expression of 10 potential candidate genes in stallions of different fertility. Frozen-thawed semen of 29 stallions was included. Each sample was classified into two groups according to pregnancy rates (PR) achieved with this semen: “good fertility” (GF; n = 17; PR ≥ 30 %) or “poor fertility” (PF; n = 12; PR <20 %). All stallions underwent a breeding soundness examination (BSE) before semen production and were only included into the semen cryopreservation program when raw semen characteristics at BSE met minimal requirements. Semen was cryopreserved following European Union regulations and all stallions met the respective health requirements. Each sample was assessed for concentration (NucleoCounter SP-100), motility (CASA), membrane functionality (SYBR-14/PI), mitochondrial membrane potential (JC-1), morphology (SpermacStain), acrosome integrity (SpermacStain), membrane integrity (HOS test) and chromatin integrity (Aniline blue). Sperm RNAs were extracted using the Direct-zol RNA Miniprep Kit (Zymo Research) and RT-qPCR was performed for each target gene. ACTB and RPL32 were included as reference genes (RGs) for normalization. For each variable of each group, mean, standard deviation and SEM were calculated. The difference in gene expression levels between the GF and PF group were analyzed using the Mann-Whitney U test and Spearman's rank correlation. Significant results were considered with p < 0.05. Sperm quality parameters did not differ significantly between the two groups except for concentration, that was significantly higher in GF (p = 0.043). In GF a positive correlation was identified for PRM1/PRM2 with r = +0.6, while PRM1/ACR (r = −0.495), PRM2/ZPBP (r = −0.645) and CRISP3/ACR (r = −0.551) were inversely correlated. In PF direct correlations were registered for PRM1/PRM2 (r = +0.629), PRM1/PRM3 (r = +0.657), PRM2/SPA17 (r = +0.685), SPA17/PLCZ1 (r = +0.786) and PRM3/ACR (r = +0.627). In the total sample (GF + PF), positive correlations were detected for PRM1/PRM2 (r = +0.625), PRM1/PRM3 (r = +0.368); PRM2/SPA17 (r = +0.465), SPA17/PLCZ1 (r = +0.637) and PLCZ1/ZAN (r = +0.587). Only two of the genes considered were differentially expressed in the 2 groups: PRM2 and PLCZ1, that were significantly (p < 0.05) overexpressed in the GF group. Stallions frozen-thawed semen with higher expression levels of PRM2 and PLCZ1 are more likely to belong to animals with a good pregnancy rate. Further studies are needed to investigate the role of sperm transcripts in male subfertility in stallions.

Magnetic Yeast Glucan Particles for Antibody-Free Separation of Viable Macrophages from Drosophila melanogaster.

Currently available methods for cell separation are generally based on fluorescent labeling using either endogenously expressed fluorescent markers or the binding of antibodies or antibody mimetics to surface antigenic epitopes. However, such modification of the target cells represents potential contamination by non-native proteins, which may affect further cell response and be outright undesirable in applications, such as cell expansion for diagnostic or therapeutic applications, including immunotherapy. We present a label- and antibody-free method for separating macrophages from living Drosophila based on their ability to preferentially phagocytose whole yeast glucan particles (GPs). Using a novel deswelling entrapment approach based on spray drying, we have successfully fabricated yeast glucan particles with the previously unachievable content of magnetic iron oxide nanoparticles while retaining their surface features responsible for phagocytosis. We demonstrate that magnetic yeast glucan particles enable macrophage separation at comparable yields to fluorescence-activated cell sorting without compromising their viability or affecting their normal function and gene expression. The use of magnetic yeast glucan particles is broadly applicable to situations where viable macrophages separated from living organisms are subsequently used for analyses, such as gene expression, metabolomics, proteomics, single-cell transcriptomics, or enzymatic activity analysis.

Abstract A019: eIF4A and PDCD4 expression in TNBC underlies sensitivity to natural product translation inhibitor

Abstract Despite recent advances in immunotherapy and adjuvant-therapy, resistance to cancer therapeutics is common and requires novel approaches. Moreover, highly aggressive triple-negative breast cancers lack effective targeted treatments. Deregulation of translation factors is associated with the increased proliferation of TNBC cells, heightened CSC activity, and poor patient outcomes. Thus targeting translation regulatory factors upregulated in TNBC is a promising approach to developing novel treatments. Pateamine A (PatA), a natural marine macrodiolide product, inhibits RNA cap-dependent translation by binding and sequestering the eukaryotic translation initiation factor eIF4A. eIF4A promotes the translation of polypurine-rich mRNA sequences, which are enriched within breast cancer oncogenes. Here we report the anticancer efficacy of novel PatA analogs we have synthesized against breast cancer cell lines. The PatA analogs exhibited selective cytotoxicity to breast and colorectal cancers compared to non-cancerous cells measured by Cell Titer-Blue viability studies. Novel analogs show greater antiproliferative potency against models of triple-negative than against human mammary epithelial cells (HMLE). To further elucidate eIF4A as a molecular target, we conducted analyses of patient tissue samples in the Cancer Genome Atlas (TCGA). Our analyses reveal the overexpression of eIF4A in tumor tissues compared to normal tissues and gene correlation studies reveal the upregulation of key co-expressed tumor drivers including beta-catenin, fibronectin, and epithelial-to-mesenchymal transition (EMT) markers such as SNAIL and SLUG. Further analyses show a downregulation of tumor suppressor protein PDCD4, an endogenous inhibitor of eIF4A, in cancer tissues. We have generated eIF4A shRNA knockdown and PDCD4 shRNA knockdown cell lines to uncover the eIF4A-PDCD4 regulatory axis across breast adenocarcinoma and colorectal adenocarcinoma cell types. We show that the loss of eIF4A attenuates the mesenchymal and stem-like phenotype of breast cancer stem cells revealed by phenotypic assays. Our results suggest that targeting translation initiator eIF4A is an effective approach against highly proliferative and metastatic breast cancer types. The presented findings underscore the potential of eIF4A as a promising molecular target and supports translational control as a viable approach against aggressive cancers. Citation Format: Alagu Subramanian, Haoran Xue, Kenneth G Hull, Daniel Romo, Joseph Taube. eIF4A and PDCD4 expression in TNBC underlies sensitivity to natural product translation inhibitor [abstract]. In: Proceedings of the AACR-NCI-EORTC Virtual International Conference on Molecular Targets and Cancer Therapeutics; 2023 Oct 11-15; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2023;22(12 Suppl):Abstract nr A019.

Abstract B097: Molecular disparities between cancer patients with normal weight and excessive body fat

Abstract Overweight and obesity, as measured by high body mass index (BMI), can have significant negative impacts on human health due to associated co-morbidities. Epidemiological data have demonstrated strong associations between overweight/obesity and 13 different types of cancer. However, the molecular features underlying the relationship between body weight and cancer remain poorly understood. In this study, we conducted a comprehensive analysis of molecular differences between patients with overweight/obesity and normal-weight patients across 14 different cancer types from The Cancer Genome Atlas (TCGA). By employing the propensity score weighting algorithm to balance confounding factors, we identified obesity-specific mutational features in multiple cancer types, such as a higher mutation burden in rectal cancer, biased mutational signatures, and overweight/obesity-specific significantly mutated genes in several cancer types. The biased mutation signatures are caused by the activity of AID/APOBEC family of cytidine deaminases and their contribution to DNA damage, ect, which suggest that obesity may contribute to the development of cancer by promoting the accumulation of specific types of mutations. Most of these biased genes have not been reported as significantly mutated genes in previous TCGA large cohort, implying these biased genes may be due to the environment of excessive body fat. We also found that differential expression genes (DEGs) and dysmethylated genes in overweight and obese tumors were predominantly upregulated and enriched in inflammatory and hormone-related pathways. These DEGs were significantly associated with survival rates in various cancer types, highlighting the impact of excessive body fat on gene expression profiles and clinical outcomes in cancer patients. Interestingly, while high BMI seemed to have a negative impact on most cancer types, it may be beneficial to endometrial cancers, as reflected by normal weight-biased mutational and gene expression patterns, suggesting the presence of an "obesity paradox" in this context. Moreover, our analysis revealed that excessive body fat significantly impacted the tumor microenvironment by altering immune cell infiltration, emphasizing the importance of understanding the complex interplay between obesity and immune response in cancer progression and treatment. Overall, our results provide the most comprehensive understanding of the molecular differences including mutational patterns, gene expression, and immune features between normal-weight and overweight/obese cancer patients. Such systematic understanding of the molecular basis for body weight disparities in multiple cancer types may provide critical insights into the future development of precision cancer therapy for cancer patients with overweight/obesity. Citation Format: Fengyuan Huang, Peng Xu, Zongliang Yue, Yuwei Song, Kaili Hu, Xinyang Zhao, Min Gao, Zechen Chong. Molecular disparities between cancer patients with normal weight and excessive body fat [abstract]. In: Proceedings of the 16th AACR Conference on the Science of Cancer Health Disparities in Racial/Ethnic Minorities and the Medically Underserved; 2023 Sep 29-Oct 2;Orlando, FL. Philadelphia (PA): AACR; Cancer Epidemiol Biomarkers Prev 2023;32(12 Suppl):Abstract nr B097.

Developmental synapse pathology triggered by maternal exposure to the herbicide glufosinate ammonium

Environmental and genetic factors influence synapse formation. Numerous animal experiments have revealed that pesticides, including herbicides, can disturb normal intracellular signals, gene expression, and individual animal behaviors. However, the mechanism underlying the adverse outcomes of pesticide exposure remains elusive. Herein, we investigated the effect of maternal exposure to the herbicide glufosinate ammonium (GLA) on offspring neuronal synapse formation in vitro. Cultured cerebral cortical neurons prepared from mouse embryos with maternal GLA exposure demonstrated impaired synapse formation induced by synaptic organizer neuroligin 1 (NLGN1)–coated beads. Conversely, the direct administration of GLA to the neuronal cultures exhibited negligible effect on the NLGN1-induced synapse formation. The comparison of the transcriptomes of cultured neurons from embryos treated with maternal GLA or vehicle and a subsequent bioinformatics analysis of differentially expressed genes (DEGs) identified “nervous system development,” including “synapse,” as the top-ranking process for downregulated DEGs in the GLA group. In addition, we detected lower densities of parvalbumin (Pvalb)-positive neurons at the postnatal developmental stage in the medial prefrontal cortex (mPFC) of offspring born to GLA–exposed dams. These results suggest that maternal GLA exposure induces synapse pathology, with alterations in the expression of genes that regulate synaptic development via an indirect pathway distinct from the effect of direct GLA action on neurons.

Gene therapy for adenosine deaminase severe combined immune deficiency-An unexpected journey of four decades.

Severe combined immune deficiency due to adenosine deaminase deficiency (ADA SCID) is an inborn error of immunity with pan-lymphopenia, due to accumulated cytotoxic adenine metabolites. ADA SCID has been treated using gene therapy with a normal human ADA gene added to autologous hematopoietic stem cells (HSC) for over 30 years. Iterative improvements in vector design, HSC processing methods, and clinical HSC transplant procedures have led nearly all ADA SCID gene therapy patients to achieve consistently beneficial immune restoration with stable engraftment of ADA gene-corrected HSC over the duration of observation (as long as 20 years). One gene therapy for ADA SCID is approved by the European Medicines Agency (EMA) in the European Union (EU) and another is being advanced to licensure in the U.S. and U.K. Despite the clear-cut benefits and safety of this curative gene and cell therapy, it remains challenging to achieve sustained availability and access, especially for rare disorders like ADA SCID.

Female Hemophilia in a Set of Triplets

Female Hemophilia In A Set Of Triplets Introduction: Hemophilia A (HA) and B (HB) are the most common inherited bleeding disorders. Gene sequencing identifies specific mutations and their associations with plasma factor levels and clinical phenotypes. Both HA and HB are X-linked recessive disorders, where the female carriers pass the gene onto their descendants. Males who inherit the mutated gene from their mothers develop the disease, whereas females are carriers due to having one normal gene, they typically do not display the characteristic features of hemophilia and have moderately low factor VIII levels of less than 40%. Female carriers may have bleeding symptoms, yet they do not experience as severe morbidities as the male hemophilia patients, due to having higher plasma factor VIII levels. Rarely female carriers can be severely affected and may have plasma factor VIII activities comparable to those with severe hemophilia. Here we are presenting a set of triplets, where the male infant and one of his sisters have severe hemophilia while the other sister is a hemophilia carrier. Methods: A set of triplets a male and two female neonates were born after 33 weeks of gestation to a healthy mother. Female neonates were dichorionic but shared the same placenta. The male infant had a circumcision at six weeks of age, which was complicated with prolonged bleeding. He was brought to the hospital and was diagnosed with severe HA. (Table.1). Infant #2 was admitted to the hospital for skin rash at age 8 weeks. She had staphylococcal scalded skin syndrome and was treated with vancomycin and cephalexin. She was noted to have prolonged bleeding from needle puncture sites. She was diagnosed with severe HA. The bleeding was controlled with tight packing. After a week of treatment her rash cleared, and she was discharged home. (Table 1). Infant #3 She was a healthy baby with no bleeding problems. (Table 1). Results and conclusions: We presented three infant-triplets-with factor VIII deficiency; infant #1 and infant #2 have severe HA. Infant #3 is an asymptomatic carrier. They all have the same mutation as their asymptomatic carrier mother. Infant #1 the male infant developed severe HA due to having the hemophilia allele on his “X” chromosome. Infant #3 a female infant is an asymptomatic carrier. She inherited the hemophilia allele on the X chromosome from her carrier mother and has an X chromosome bearing the normal allele, thus is able to produce factor VIII of 40%. Infant #2 the other female infant had severe HA, with a factor VIII activity of &amp;lt;0.3%. Female hemophilia is rare occurrence. Hemophilia treatment center (HTT) population profile (PP) reported that of the 1,676 females attending to HTC, 47.6% of them had less than 40% factor level, 0.48% of them had severe, 1.4% had moderate and 17.9% had mild hemophilia[Haemophilia. 2021:27(6):1037-1044. Doi:10.1111}. This can occur, in the setting of monosomy X (Turner syndrome-XO), due to having one X chromosome carrying the hemophilic gene or in the setting of compound heterozygosity: the female product of a father with hemophilia and the mother being a carrier. Similarly, hemizygosity of the hemophilic gene on both parents would result in hemophilia in their daughters. Another mechanism that plays a role in the pathogenesis of female hemophilia is the “X chromosome inactivation (XCI). This has been described as the most common cause of female hemophilia. One of the two X chromosomes in females is randomly and permanently inactivated in cells to ensure the balance with one X chromosome of males and limit the overproduction of potentially toxic gene proteins. The degree of inactivation determines the plasma levels of the gene products, in hemophilia the factor levels. The chromosome analysis of infant #2 revealed normal 46XX. Structural chromosomal abnormality is ruled out as she did not have Turner syndrome “XO”, or “45XY” with androgen insensitivity syndrome, nor did she have compound heterozygosity or hemizygosity of the X chromosome. The father had normal coagulation studies and factor VIII activity. Therefore, we believe infant #2 had extremely skewed XCI and the inactivation is skewed in favor of hemophilic X chromosome. This mechanism resulted in severe HA in this infant. The infants are now seven months old and have no bleeding problems, as infant #1 and #2 are on prophylaxis with emicizumab (Hemlibra), they both have 1% factor activity by chromogenic assay and do not have inhibitors. (Table 1)

Molecular Subtypes in BCR::ABL1-Positive ALL Are Defined By Cellular Origins and Cooperating Genomic Events

Two developmental trajectories are acknowledged in BCR::ABL1-positive acute lymphoblastic leukemia (ALL) as distinct diagnostic entities by the ICC classification: ‘lymphoid-only’ with BCR::ABL1 restricted to the leukemic B precursor compartment vs. ‘multilineage’ with BCR::ABL1 involvement also in other hematopoietic lineages. Diagnostic standards for establishing this distinction are lacking and associated biological and clinical features are insufficiently characterized. To establish developmental trajectories, biological phenotypes, and clinical impact we analyzed n=277 BCR::ABL1-positive ALL patients (age: 2-84 years, median: 46) including our GMALL reference data set (n=113) and two independent validation cohorts (MLL: n=61; St Jude's: n=103; Gu Z, et al. Nat Genet. 2019). Unsupervised gene expression analysis (RNA-Seq: n=277) identified two major gene expression clusters with two further sub-clusters each (Figure A). This clustering was confirmed by a machine learning classifier trained on the GMALL data set which achieved similar grouping of samples in the external validation cohorts. BCR:: ABL1-FISH on FACS-sorted bone marrow/peripheral blood samples revealed the presence of BCR::ABL1 in leukemic as well as myeloid cells in n=18/18 samples from the 1 st major cluster (termed 'multilineage'). In the 2 nd major cluster ('lymphoid'), BCR::ABL1 was restricted to the lymphoid lineage in n=13/16 samples (p&amp;lt;0.001), with n=3/16 cases harboring BCR::ABL1-positive myeloid cells at lower frequencies compared to the multilineage cluster. In each cluster, two patients also harbored BCR::ABL1-positive mature B cells. T cells remained BCR::ABL1-negative. Diagnostic FACS data and analysis of proximity to normal human lymphoid gene expression confirmed more frequent myeloid co-expression and higher proximity to normal pro-B cells in the multilineage cluster, whereas lymphoid cases had increased lymphoid surface marker expression and a stronger proximity to normal pre-B I cells. Genomic profiling using WGS/SNParrays (n=160) revealed significant enrichment for genomic events in the four gene expression sub-clusters: focal deletions comprising exons 1 and 2 of HBS1-like translational GTPase ( HBS1L) or monosomy 7 were strongly enriched in the two multilineage sub-clusters ('delHBS1L'; n=20/27 vs. n=3/133 in remaining cohort or 'del7'; n=16/25 vs. n=10/135; p&amp;lt;0.001). Remarkably, a novel alternative HBS1L transcript with a putative TSS in intron 3 was highly expressed in both multilineage sub-clusters but not in BCR::ABL1 negative ALL cases or healthy B lymphoid progenitors, suggesting this transcript as novel cooperating event in multilineage BCR::ABL1-positive ALL. One lymphoid sub-cluster was enriched for homozygous deletions in IKZF1 ('IKZF1'; n=11/55 vs. n=4/105; p=0.008), whereas the other was enriched for homozygous CDKN2A/B deletions (n=21/53 vs. n=3/107; p&amp;lt;0.001) and PAX5 deletions (n=33/53 vs n=26/107; p&amp;lt;0.001; 'CDKN2A/PAX5'). Hyperdiploid karyotypes were exclusive to the lymphoid main cluster, mostly in CDKN2A/PAX5. We analyzed the clinical implications of these newly established BCR::ABL1-positive ALL subtypes in our homogenously treated GMALL adult patient cohort (n=98, first diagnosis 2014-2021) including TKI treatment combined with age-adapted chemotherapy, MRD monitoring and allo-SCT in 1 st CR (n=84/98, 86%). Overall survival (OS) at 3 years was uniformly high in multilineage and lymphoid cases (70%±6% vs. 70%±8%; p=0.890; Figure B). However, analysis of the sub-clusters revealed an inferior 3-years OS for the IKZF1 -/- enriched cluster in contrast to excellent outcome in hyperdiploid cases and intermediate outcomes in the remaining sub-clusters (Figure B). These data highlight that transcriptomic signatures in BCR::ABL1-positive ALL are driven by developmental disease origins (‘lymphoid’ vs. ‘multilineage‘) and specific patterns of corresponding genomic events. Novel molecular subtypes of BCR::ABL1-positive ALL have distinct outcomes in the context of current GMALL treatment protocols. Gene-expression based subtype definitions enable classification of BCR::ABL1-positive ALL according to ICC definitions based on RNA-Seq data alone. These definitions have been implemented in ALLCatchR - our freely available tool for ALL subtype allocation - to facilitate validation and application in clinical diagnostics.

Label-free identification of DNA single-base mutation for clinical diagnostics based on plasmonic SiNW@AgNP arrays

Surface-enhanced Raman spectroscopy (SERS) has exhibited great potential in deoxyribonucleic acid (DNA) analysis. Rapid and label-free identification of single-base mutation in DNA is still limited by the sensitivity and reproducibility of SERS substrates. In this work, we develope plasmonic SiNW arrays decorated with Ag nanoparticles (SiNW@AgNP arrays) via hexadecyltrimethylammonium bromide (CTAB)-assisted galvanic reaction to address these challenges, which show excellent sensitivity and reproducibility. Under optimized experimental conditions, plasmonic SiNW@AgNP arrays enable detection of DNA bases of adenine (A), guanine (G), cytosine (C), and thymine (T) in solution at concentration down to pM-μM range, and multiple detection ability with mixture of DNA bases. More significantly, our study demonstrates the SiNW@AgNP arrays may serve as excellent SERS substrates in discriminating DNA single-base mutation between sickle cell anemia gene and normal hemoglobin gene in serum samples, which can be exploited in a wide range of plasmonic enhanced sensing applications for a fast, real-time, label-free and cost-effective clinical diagnosis.

The clinical, imaging, pathological and genetic landscape of bottom-of-sulcus dysplasia.

Bottom-of-sulcus dysplasia (BOSD) is increasingly recognized as a cause of drug-resistant, surgically-remediable, focal epilepsy, often in seemingly MRI-negative patients. We describe the clinical manifestations, morphological features, localization patterns and genetics of BOSD, with the aims of improving management and understanding pathogenesis. We studied 85 patients with BOSD diagnosed between 2005-2022. Presenting seizure and EEG characteristics, clinical course, genetic findings and treatment response were obtained from medical records. MRI (3 T) and 18F-FDG-PET scans were reviewed systematically for BOSD morphology and metabolism. Histopathological analysis and tissue genetic testing were performed in 64 operated patients. BOSD locations were transposed to common imaging space to study anatomical location, functional network localization and relationship to normal MTOR gene expression. All patients presented with stereotyped focal seizures with rapidly escalating frequency, prompting hospitalization in 48%. Despite 42% patients having seizure remissions, usually with sodium channel blocking medications, most eventually became drug-resistant and underwent surgery (86% seizure-free). Prior developmental delay was uncommon but intellectual, language and executive dysfunction were present in 24%, 48% and 29% when assessed preoperatively, low intellect being associated with greater epilepsy duration. BOSDs were missed on initial MRI in 68%, being ultimately recognized following repeat MRI, 18F-FDG-PET or image postprocessing. MRI features were grey-white junction blurring (100%), cortical thickening (91%), transmantle band (62%), increased cortical T1 signal (46%) and increased subcortical FLAIR signal (26%). BOSD hypometabolism was present on 18F-FDG-PET in 99%. Additional areas of cortical malformation or grey matter heterotopia were present in eight patients. BOSDs predominated in frontal and pericentral cortex and related functional networks, mostly sparing temporal and occipital cortex, and limbic and visual networks. Genetic testing yielded pathogenic mTOR pathway variants in 63% patients, including somatic MTOR variants in 47% operated patients and germline DEPDC5 or NPRL3 variants in 73% patients with familial focal epilepsy. BOSDs tended to occur in regions where the healthy brain normally shows lower MTOR expression, suggesting these regions may be more vulnerable to upregulation of MTOR activity. Consistent with the existing literature, these results highlight (i) clinical features raising suspicion of BOSD; (ii) the role of somatic and germline mTOR pathway variants in patients with sporadic and familial focal epilepsy associated with BOSD; and (iii) the role of 18F-FDG-PET alongside high-field MRI in detecting subtle BOSD. The anatomical and functional distribution of BOSDs likely explain their seizure, EEG and cognitive manifestations and may relate to relative MTOR expression.

Proteostasis governs differential temperature sensitivity across embryonic cell types

Embryonic development is remarkably robust, but temperature stress can degrade its ability to generate animals with invariant anatomy. Phenotypes associated with environmental stress suggest that some cell types are more sensitive to stress than others, but the basis of this sensitivity is unknown. Here, we characterize hundreds of individual zebrafish embryos under temperature stress using whole-animal single-cell RNA sequencing (RNA-seq) to identify cell types and molecular programs driving phenotypic variability. We find that temperature perturbs the normal proportions and gene expression programs of numerous cell types and also introduces asynchrony in developmental timing. The notochord is particularly sensitive to temperature, which we map to a specialized cell type: sheath cells. These cells accumulate misfolded protein at elevated temperature, leading to a cascading structural failure of the notochord and anatomic defects. Our study demonstrates that whole-animal single-cell RNA-seq can identify mechanisms for developmental robustness and pinpoint cell types that constitute key failure points.

Effect of microgravity on mammalian embryo development evaluated at the International Space Station

Mammalian embryos differentiate into the inner cell mass (ICM) and trophectoderm at the 8-16 cell stage. The ICM forms a single cluster that develops into a single fetus. However, the factors that determine differentiation and single cluster formation are unknown. Here we investigated whether embryos could develop normally without gravity. As the embryos cannot be handled by an untrained astronaut, a new device was developed for this purpose. Using this device, two-cell frozen mouse embryos launched to the International Space Station were thawed and cultured by the astronauts under microgravity for 4days. The embryos cultured under microgravity conditions developed into blastocysts with normal cell numbers, ICM, trophectoderm, and gene expression profiles similar to those cultured under artificial-1g control on the International Space Station and ground-1g control, which clearly demonstrated that gravity had no significant effect on the blastocyst formation and initial differentiation of mammalian embryos.

Altered Gene Expression Within the Renin-Angiotensin System in Normal Aging and Dementia.

The renin-angiotensin system (RAS) is dysregulated in Alzheimer's disease (AD). In this study, we have explored the hypothesis that an -age--related imbalance in brain RAS is a trigger for RAS dysregulation in AD. We characterized RAS gene expression in the frontal cortex from (i) a cohort of normal aging (n = 99, age range = 19-96 years) and (ii) a case-control cohort (n = 209) including AD (n = 66), mixed dementia (VaD + AD; n = 50), pure vascular dementia (VaD; n = 42), and age-matched controls (n = 51). The AD, mixed dementia, and age-matched controls were further stratified by Braak tangle stage (BS): BS0-II (n = 48), BSIII-IV (n = 44), and BSV-VI (n = 85). Gene expression was calculated by quantitative PCR (qPCR) for ACE1, AGTR1, AGTR2, ACE2, LNPEP, and MAS1 using the 2-∆∆Cq method, after adjustment for reference genes (RPL13 and UBE2D2) and cell-specific calibrator genes (NEUN, GFAP, PECAM). ACE1 and AGTR1, markers of classical RAS signaling, and AGTR2 gene expression were elevated in normal aging and gene expression in markers of protective downstream regulatory RAS signaling, including ACE2, MAS1, and LNPEP, were unchanged. In AD and mixed dementia, AGTR1 and AGTR2 gene expression were elevated in BSIII-IV and BSV-VI, respectively. MAS1 gene expression was reduced at BSV-VI and was inversely related to parenchymal Aβ and tau load. LNPEP gene expression was specifically elevated in VaD. These data provide novel insights into RAS signaling in normal aging and dementia.

Epigenetic Modulators as Therapeutic Agents in Cancer.

Epigenetics play a crucial role in gene regulation and cellular processes. Most importantly, its dysregulation can contribute to the development of tumors. Epigenetic modifications, such as DNA methylation and histone acetylation, are reversible processes that can be utilized as targets for therapeutic intervention. DNA methylation inhibitors disrupt DNA methylation patterns by inhibiting DNA methyltransferases. Such inhibitors can restore normal gene expression patterns, and they can be effective against various forms of cancer. Histone deacetylase inhibitors increase histone acetylation levels, leading to altered gene expressions. Like DNA methylation inhibitors, histone methyltransferase inhibitors target molecules involved in histone methylation. Bromodomain and extra-terminal domain inhibitors target proteins involved in gene expression. They can be effective by inhibiting oncogene expression and inducing anti-proliferative effects seen in cancer. Understanding epigenetic modifications and utilizing epigenetic inhibitors will offer new possibilities for cancer research.

FRI476 Asymptomatic Thyrotropin-secreting Pituitary Microadenoma With Biochemical Secondary Hyperthyroidism: A Diagnostic Conundrum

Abstract Disclosure: G. Al-Naqeeb: None. R. Ghosh: None. P. Veeraraghavan: None. C. Cochran: None. J. Klubo-Gwiezdzinska: None. S. Gubbi: None. Background: Thyrotropin (TSH)-secreting pituitary adenomas (TSHAs) are often macroadenomas (&amp;gt;1 cm), and clinically present with secondary hyperthyroidism (SH), but clinically silent microTSHAs (&amp;lt;1 cm) with only biochemical SH pose a diagnostic challenge. Clinical Case: A 49-year-old, healthy male was referred to our center for evaluation of abnormal thyroid function tests (TFTs). Immunoassay (IA) measurements at an outside hospital had identified elevated free T4 (FT4), and an inappropriately normal TSH, and a pituitary magnetic resonance imaging (MRI) had noted an 8mm x 7mm non-invasive adenoma. The patient denied hyperthyroidism symptoms, medication/herbal supplement intake, radiation exposure, headaches, vision changes, and thyroid/pituitary disorders, or abnormal TFTs in relatives. Physical exam revealed no signs and symptoms of hyperthyroidism. No thyromegaly or cervical lymphadenopathy was felt. IA profile at our institute was consistent with an SH pattern [TSH: 3.32 mIU/L (NL: 0.4 - 4.7); FT4: 3 ng/dL (NL: 0.9 - 1.7); total T4: 14.5 mcg/dL (NL: 4.5 - 11.7); triiodothyronine: 266.6 ng/dL (NL: 80 - 200)], with similar values on repeat measurement. Equilibrium dialysis technique also yielded an elevated FT4 value. TSH-receptor, anti-thyroid peroxidase, and anti-thyroglobulin (Tg) antibody (Ab) titers, serum Tg, and thyroid binding globulin levels were normal. Dilution studies ruled out heterophile Ab interference. Familial dysalbuminemic hyperthyroxinemia was unlikely given the elevated FT4 values. Genetic testing revealed a normal THRB gene, thereby ruling out thyroid hormone resistance. THRA gene was not tested due to absence of behavioral or hearing issues. Markers of hyperthyroidism, including angiotensin converting enzyme, carotene, apolipoproteins, and osteocalcin levels were normal, except for an elevated sex hormone binding globulin of 104 nmol/L (NL: 11 - 78). Biochemical evaluation of other pituitary axes was normal. However, an elevated alpha sub-unit fraction (ASF) was noted on two separate measurements. Sonogram revealed a heterogeneous, hypervascular thyroid. Bone densitometry, echocardiogram, and Holter monitor studies were normal. The combination of biochemical SH, elevated ASF, pituitary adenoma, and exclusion of other etiologies causing SH pattern established the diagnosis of TSHA. An active surveillance approach was implemented. A pituitary MRI done 18 months later showed minimal (2mm in one axis) increase in the adenoma size, and the patient continued to demonstrate biochemical SH but remained asymptomatic. Conclusion: MicroTSHAs can present with asymptomatic SH. In this situation, excluding other biochemical SH etiologies is crucial to prevent unnecessary invasive procedures. But once TSHA diagnosis is established, due to the risk of tumor growth leading to symptomatic SH or mass effects, active surveillance is warranted. Presentation: Friday, June 16, 2023

THU459 Hypophosphatasia With Normal ALPL Gene Test; A Case Report

Abstract Disclosure: K. Alkwatli: None. L.Z. Khan: None. Introduction: Hypophosphatasia is a rare hereditary disorder caused by loss of tissue nonspecific alkaline phosphatase activity, an essential enzyme in phosphate metabolism. Severe cases present perinatally and in early childhood. Mild cases can present in adulthood and can be misdiagnosed. We present an interesting patient who was clinically diagnosed with hypophosphatasia despite negative molecular genetic testing. Case presentation: 40 years-old female with a history of fibromyalgia and multiple bone fractures presented to the clinic with a long-standing history of multiple cavities, severe fatigue, headaches, muscle cramps, and arthralgia. The patient had numerous fractures throughout her life that started at age five without significant trauma, including wrist, forearm, and metatarsal bones. Her family history was negative. Laboratory testing showed low alkaline phosphatase levels on multiple occasions, 28,29,30 U/L (reference range 34-123). Her vitamin B6 was elevated at 309 nmol/L (20-125). Her zinc, calcium, vitamin D, and liver function tests were all within normal limits. Bone density scan showed a Z score of -1.0 at the lumbar spine and a Z score of -0.3 at the left femoral neck. Molecular genetic testing of the ALPL gene was negative. Hypophosphatasia was diagnosed based on her clinical and lab assessments, and she is being treated with Asfotase alfa. Her clinical course is yet to be determined. Discussion: ALPL gene is responsible for encoding the tissue nonspecific alkaline phosphatase (TNALP), the main enzyme affected in patients with hypophosphatasia. TNALP degrades inorganic pyrophosphate (PPi) to inorganic phosphate (Pi), an essential element for bone mineralization. It also facilitates vitamin B6 to cross the cell membranes. Therefore, loss of function mutations of the ALPL gene cause accumulation of PPi, which impairs the calcium-phosphate hemostasis and causes damage to multiple organs. In addition, the accumulation of the active form of vitamin B6 extracellularly can result in neurological damage.Patients present with a broad spectrum of musculoskeletal, renal, dental, and neurological symptoms. The variability of the phenotypic magnitudes is thought to be due to heterogeneity in the ALPL gene mutations. Over 400s of ALPL gene mutations have been identified in patients with hypophosphatasia. Novel variants are increasingly being reported.Negative genetic testing of the ALPL gene can still be seen in patients with hypophosphatasia due to either a variant that is undetectable by current testing methodology or if the causative gene/s are not on the ALPL gene panel. Conclusion: Genetic testing is helpful in confirming the disease and providing genetic counseling; however, treatment should still be considered if patients have sufficient clinical and laboratory findings. Presentation: Thursday, June 15, 2023

THU432 Severe Osteoporosis Associated With A Novel Plastin 3 (PLS3) Mutation

Abstract Disclosure: A. Ibrahim: None. L. Esper: None. S.M. Gandhi: None. E.S. Nylen: None. Early Onset Osteoporosis (EOOP) affects children and young adults and can be caused by endocrinopathies, chronic medical conditions, nutritional deficiencies, and/or genetic mutations. The most common inherited cause is osteogenesis imperfecta(OI), and a rare variant linked to OI includes a defect in the X chromosomal gene encoding Plastin 3 (PLS3). In our case, a 34-year-old male was referred for severe osteoporosis. He had been diagnosed with osteopenia at age 21. At age 24, imaging revealed multiple vertebral compression fractures and DXA scan confirmed severe osteoporosis. He required L3-L5 kyphoplasty and started on oral bisphosphonates for presumed OI despite negative gene mutation (i.e., COL1A1/COL1A2). He continued treatment for only 2 years. 8 years later, he had persistent osteoporosis (DXA L2-4 Z-score -3.6, LFN Z-score -3.2). He had no evidence of kyphosis or scoliosis and no extraskeletal manifestations such as blue-grey sclera or any dysmorphic features. The workup was negative for secondary causes of EOOP and he denied a family history of osteoporosis. Labs showed normal 24-hour urine calcium, bone markers, and bone Alkaline Phosphatase. Karyotyping showed normal XY and gene analysis was again negative for OI. He started treatment with intravenous Zoledronic acid, however, had a 2-year gap between doses initially causing BMD to drop by 6.5% in L2-L4 (Z -4.1) and by 5.7% in LFN (Z -3.1). Subsequently, he received a total of 5 doses of Zoledronic acid to date and had no further fractures. DXA significantly improved by 10.4% in L2-4 (Z -2.6), and 16.3% in LFN (Z -2.0). Repeated expanded gene testing showed a novel PLS3 hemizygous mutation (deletion at site C.234_237+10del of Exon 3). This case demonstrates a novel PLS3 mutation variant to be a plausible cause of adulthood EOOP which was responsive to bisphosphonate treatment. PLS3 is an F-actin binding and bundling protein involved in cytoskeletal remodeling and may have roles in calcium homeostasis, cell migration, endocytosis, DNA repair, and membrane trafficking. Currently, less than 30 loss of function variants of PLS3 have been identified and all lead to severe osteoporosis- particularly in children. However, our patient’s known onset of bone loss occurred much later- in his twenties and he had no evidence of extra-skeletal manifestations. The cause for such late presentation is unclear but may be attributed to this specific variant of PLS3. This case highlights that PLS3 mutation should be on the differential for EOOP even in early adulthood. Our patient’s BMD has markedly improved with bisphosphonates and he sustained no further fractures after starting therapy. Timely diagnosis and earlier detection will allow for appropriate genetic counseling, earlier screening, and treatment of affected family members. Presentation: Thursday, June 15, 2023

Evaluation of potential reference genes in the biting midge Culicoides sonorensis for real-time quantitative PCR analyses

Studies examining differentially expressed genes and gene silencing by RNA interference (RNAi) require a set of stably expressed reference genes for accurate normalization. The biting midge Culicoides sonorensis is an important vector of livestock pathogens and is often used as a model species for biting midge research. Here, we examine the stable expression of six candidate reference genes in C. sonorensis: actin, β-tubulin, glyceraldehyde 3-phosphate dehydrogenase (GAPDH), ribosomal protein subunit (RPS) 18, vacuolar ATPase subunit A (VhaA), and elongation factor 1-beta (EF1b). Gene expression was assessed under seven conditions, including cells treated with double-stranded RNA (dsRNA), 3rd and 4th instar larvae treated with dsRNA, six developmental stages, four adult female body parts or tissue groups, and females injected with bluetongue virus or vesicular stomatitis virus. Stable gene expression was assessed using RefFinder, NormFinder, geNorm, and BestKeeper. The ranked results for each analysis tool under each condition and a comprehensive ranking for each condition are presented. The data show that optimal reference genes vary between conditions and that just two reference genes were necessary for each condition. These findings provide reference genes for use under these conditions in future studies using real-time quantitative PCR to evaluate gene expression in C. sonorensis.