Benjamin W. Darbro

Development and translational imaging of a TP53 porcine tumorigenesis model

Cancer is the second deadliest disease in the United States, necessitating improvements in tumor diagnosis and treatment. Current model systems of cancer are informative, but translating promising imaging approaches and therapies to clinical practice has been challenging. In particular, the lack of a large-animal model that accurately mimics human cancer has been a major barrier to the development of effective diagnostic tools along with surgical and therapeutic interventions. Here, we developed a genetically modified porcine model of cancer in which animals express a mutation in TP53 (which encodes p53) that is orthologous to one commonly found in humans (R175H in people, R167H in pigs). TP53(R167H/R167H) mutant pigs primarily developed lymphomas and osteogenic tumors, recapitulating the tumor types observed in mice and humans expressing orthologous TP53 mutant alleles. CT and MRI imaging data effectively detected developing tumors, which were validated by histopathological evaluation after necropsy. Molecular genetic analyses confirmed that these animals expressed the R167H mutant p53, and evaluation of tumors revealed characteristic chromosomal instability. Together, these results demonstrated that TP53(R167H/R167H) pigs represent a large-animal tumor model that replicates the human condition. Our data further suggest that this model will be uniquely suited for developing clinically relevant, noninvasive imaging approaches to facilitate earlier detection, diagnosis, and treatment of human cancers.

Disruption of the non-canonical Wnt gene PRICKLE2 leads to autism-like behaviors with evidence for hippocampal synaptic dysfunction

Autism spectrum disorders (ASDs) have been suggested to arise from abnormalities in the canonical and non-canonical Wnt signaling pathways. However, a direct connection between a human variant in a Wnt pathway gene and ASD-relevant brain pathology has not been established. Prickle2 (Pk2) is a post-synaptic non-canonical Wnt signaling protein shown to interact with post-synaptic density 95 (PSD-95). Here, we show that mice with disruption in Prickle2 display behavioral abnormalities including altered social interaction, learning abnormalities and behavioral inflexibility. Prickle2 disruption in mouse hippocampal neurons led to reductions in dendrite branching, synapse number and PSD size. Consistent with these findings, Prickle2 null neurons show decreased frequency and size of spontaneous miniature synaptic currents. These behavioral and physiological abnormalities in Prickle2 disrupted mice are consistent with ASD-like phenotypes present in other mouse models of ASDs. In 384 individuals with autism, we identified two with distinct, heterozygous, rare, non-synonymous PRICKLE2 variants (p.E8Q and p.V153I) that were shared by their affected siblings and inherited paternally. Unlike wild-type PRICKLE2, the PRICKLE2 variants found in ASD patients exhibit deficits in morphological and electrophysiological assays. These data suggest that these PRICKLE2 variants cause a critical loss of PRICKLE2 function. The data presented here provide new insight into the biological roles of Prickle2, its behavioral importance, and suggest disruptions in non-canonical Wnt genes such as PRICKLE2 may contribute to synaptic abnormalities underlying ASDs.

Eating disorder predisposition is associated with ESRRA and HDAC4 mutations

Anorexia nervosa and bulimia nervosa are common and severe eating disorders (EDs) of unknown etiology. Although genetic factors have been implicated in the psychopathology of EDs, a clear biological pathway has not been delineated. DNA from two large families affected by EDs was collected, and mutations segregating with illness were identified by whole-genome sequencing following linkage mapping or by whole-exome sequencing. In the first family, analysis of twenty members across three generations identified a rare missense mutation in the estrogen-related receptor α (ESRRA) gene that segregated with illness. In the second family, analysis of eight members across four generations identified a missense mutation in the histone deacetylase 4 (HDAC4) gene that segregated with illness. ESRRA and HDAC4 were determined to interact both in vitro in HeLa cells and in vivo in mouse cortex. Transcriptional analysis revealed that HDAC4 potently represses the expression of known ESRRA-induced target genes. Biochemical analysis of candidate mutations revealed that the identified ESRRA mutation decreased its transcriptional activity, while the HDAC4 mutation increased transcriptional repression of ESRRA. Our findings suggest that mutations that result in decreased ESRRA activity increase the risk of developing EDs.

The heritability of metabolite concentrations in stored human red blood cells.

The degeneration of red blood cells (RBCs) during storage is a major issue in transfusion medicine. Family studies in the 1960s established the heritability of the RBC storage lesion based on poststorage adenosine triphosphate (ATP) concentrations. However, this critical discovery has not been further explored. In a classic twin study we confirmed the heritability of poststorage ATP concentrations and established the heritability of many other RBC metabolites.

Seizures Are Regulated by Ubiquitin-specific Peptidase 9 X-linked (USP9X), a De-Ubiquitinase

Epilepsy is a common disabling disease with complex, multifactorial genetic and environmental etiology. The small fraction of epilepsies subject to Mendelian inheritance offers key insight into epilepsy disease mechanisms; and pathologies brought on by mutations in a single gene can point the way to generalizable therapeutic strategies. Mutations in the PRICKLE genes can cause seizures in humans, zebrafish, mice, and flies, suggesting the seizure-suppression pathway is evolutionarily conserved. This pathway has never been targeted for novel anti-seizure treatments. Here, the mammalian PRICKLE-interactome was defined, identifying prickle-interacting proteins that localize to synapses and a novel interacting partner, USP9X, a substrate-specific de-ubiquitinase. PRICKLE and USP9X interact through their carboxy-termini; and USP9X de-ubiquitinates PRICKLE, protecting it from proteasomal degradation. In forebrain neurons of mice, USP9X deficiency reduced levels of Prickle2 protein. Genetic analysis suggests the same pathway regulates Prickle-mediated seizures. The seizure phenotype was suppressed in prickle mutant flies by the small-molecule USP9X inhibitor, Degrasyn/WP1130, or by reducing the dose of fat facets a USP9X orthologue. USP9X mutations were identified by resequencing a cohort of patients with epileptic encephalopathy, one patient harbored a de novo missense mutation and another a novel coding mutation. Both USP9X variants were outside the PRICKLE-interacting domain. These findings demonstrate that USP9X inhibition can suppress prickle-mediated seizure activity, and that USP9X variants may predispose to seizures. These studies point to a new target for anti-seizure therapy and illustrate the translational power of studying diseases in species across the evolutionary spectrum.

PRICKLE1 Interaction with SYNAPSIN I Reveals a Role in Autism Spectrum Disorders

The frequent comorbidity of Autism Spectrum Disorders (ASDs) with epilepsy suggests a shared underlying genetic susceptibility; several genes, when mutated, can contribute to both disorders. Recently, PRICKLE1 missense mutations were found to segregate with ASD. However, the mechanism by which mutations in this gene might contribute to ASD is unknown. To elucidate the role of PRICKLE1 in ASDs, we carried out studies in Prickle1+/− mice and Drosophila, yeast, and neuronal cell lines. We show that mice with Prickle1 mutations exhibit ASD-like behaviors. To find proteins that interact with PRICKLE1 in the central nervous system, we performed a yeast two-hybrid screen with a human brain cDNA library and isolated a peptide with homology to SYNAPSIN I (SYN1), a protein involved in synaptogenesis, synaptic vesicle formation, and regulation of neurotransmitter release. Endogenous Prickle1 and Syn1 co-localize in neurons and physically interact via the SYN1 region mutated in ASD and epilepsy. Finally, a mutation in PRICKLE1 disrupts its ability to increase the size of dense-core vesicles in PC12 cells. Taken together, these findings suggest PRICKLE1 mutations contribute to ASD by disrupting the interaction with SYN1 and regulation of synaptic vesicles.

The heritability of hemolysis in stored human red blood cells

The transfusion of red blood cells (RBCs) with maximum therapeutic efficacy is a major goal in transfusion medicine. One of the criteria used in determining stored RBC quality is end‐of‐storage hemolysis. Between donors, a wide range of hemolysis is observed under identical storage conditions. Here, a potential mechanism for this wide range is investigated. We hypothesize that the magnitude of hemolysis is a heritable trait. Also, we investigated correlations between hemolysis and RBC metabolites; this will establish pathways influencing hemolysis as future targets for genetic analysis.

Mutations in Extracellular Matrix Genes NID1 and LAMC1 Cause Autosomal Dominant Dandy–Walker Malformation and Occipital Cephaloceles

We performed whole‐exome sequencing of a family with autosomal dominant Dandy–Walker malformation and occipital cephaloceles and detected a mutation in the extracellular matrix (ECM) protein‐encoding gene NID1. In a second family, protein interaction network analysis identified a mutation in LAMC1, which encodes a NID1‐binding partner. Structural modeling of the NID1–LAMC1 complex demonstrated that each mutation disrupts the interaction. These findings implicate the ECM in the pathogenesis of Dandy–Walker spectrum disorders.

A novel porcine model of ataxia telangiectasia reproduces neurological features and motor deficits of human disease

Ataxia telangiectasia (AT) is a progressive multisystem disorder caused by mutations in the AT-mutated (ATM) gene. AT is a neurodegenerative disease primarily characterized by cerebellar degeneration in children leading to motor impairment. The disease progresses with other clinical manifestations including oculocutaneous telangiectasia, immune disorders, increased susceptibly to cancer and respiratory infections. Although genetic investigations and physiological models have established the linkage of ATM with AT onset, the mechanisms linking ATM to neurodegeneration remain undetermined, hindering therapeutic development. Several murine models of AT have been successfully generated showing some of the clinical manifestations of the disease, however they do not fully recapitulate the hallmark neurological phenotype, thus highlighting the need for a more suitable animal model. We engineered a novel porcine model of AT to better phenocopy the disease and bridge the gap between human and current animal models. The initial characterization of AT pigs revealed early cerebellar lesions including loss of Purkinje cells (PCs) and altered cytoarchitecture suggesting a developmental etiology for AT and could advocate for early therapies for AT patients. In addition, similar to patients, AT pigs show growth retardation and develop motor deficit phenotypes. By using the porcine system to model human AT, we established the first animal model showing PC loss and motor features of the human disease. The novel AT pig provides new opportunities to unmask functions and roles of ATM in AT disease and in physiological conditions.

A Gene Implicated in Activation of Retinoic Acid Receptor Targets Is a Novel Renal Agenesis Gene in Humans

Renal agenesis is a devastating birth defect, and although genes encoding retinoic acid signaling components have been shown to be important for renal... Renal agenesis (RA) is one of the more extreme examples of congenital anomalies of the kidney and urinary tract (CAKUT). Bilateral renal agenesis is almost invariably fatal at birth, and unilateral renal agenesis can lead to future health issues including end-stage renal disease. Genetic investigations have identified several gene variants that cause RA, including EYA1, LHX1, and WT1. However, whereas compound null mutations of genes encoding α and γ retinoic acid receptors (RARs) cause RA in mice, to date there have been no reports of variants in RAR genes causing RA in humans. In this study, we carried out whole exome sequence analysis of two families showing inheritance of an RA phenotype, and in both identified a single candidate gene, GREB1L. Analysis of a zebrafish greb1l loss-of-function mutant revealed defects in the pronephric kidney just prior to death, and F0 CRISPR/Cas9 mutagenesis of Greb1l in the mouse revealed kidney agenesis phenotypes, implicating Greb1l in this disorder. GREB1L resides in a chromatin complex with RAR members, and our data implicate GREB1L as a coactivator for RARs. This study is the first to associate a component of the RAR pathway with renal agenesis in humans.