Publications by authors named "Benjamin W Darbro"

50 Publications

Sequential genetic testing of living related donors for inherited renal disease to promote informed choice and enhance safety of living donation.

Transpl Int 2021 Oct 10. Epub 2021 Oct 10.

Departments of Internal Medicine, University of Iowa, Iowa City, IA, USA.

Living kidney donors (LKDs) with a family history of renal disease are at risk of kidney disease as compared to LKDs without such history suggesting that some LKDs may be pre-symptomatic for monogenic kidney disease. LKDs with related transplant candidates whose kidney disease was considered genetic in origin were selected for genetic testing. In each case, the transplant candidate was first tested to verify the genetic diagnosis. A genetic diagnosis was confirmed in 12 of 24 transplant candidates (ADPKD-PKD1: 6, ALPORT-COL4A3: 2, ALPORT-COL4A5: 1: nephronophthisis-SDCCAG8: 1; CAKUT-HNF1B and ADTKD-MUC1: 1 each) and 2 had variants of unknown significance (VUS) in phenotype-relevant genes. Focused genetic testing was then done in 20 of 34 LKDs. 12 LKDs screened negative for the familial variant and were permitted to donate; 7 screened positive and were counseled against donation. One, the heterozygous carrier of a recessive disorder was also cleared. 6 of 7 LKDs with a family history of ADPKD were under 30 yr and in 5, by excluding ADPKD, allowed donation to safely proceed. The inclusion of genetic testing clarified the diagnosis in recipient candidates, improving safety or informed decision making in LKDs.
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http://dx.doi.org/10.1111/tri.14133DOI Listing
October 2021

RABL6A Promotes Pancreatic Neuroendocrine Tumor Angiogenesis and Progression In Vivo.

Biomedicines 2021 Jun 2;9(6). Epub 2021 Jun 2.

Department of Neuroscience and Pharmacology, The University of Iowa, Iowa City, IA 52242, USA.

Pancreatic neuroendocrine tumors (pNETs) are difficult-to-treat neoplasms whose incidence is rising. Greater understanding of pNET pathogenesis is needed to identify new biomarkers and targets for improved therapy. RABL6A, a novel oncogenic GTPase, is highly expressed in patient pNETs and required for pNET cell proliferation and survival in vitro. Here, we investigated the role of RABL6A in pNET progression in vivo using a well-established model of the disease. RIP-Tag2 (RT2) mice develop functional pNETs (insulinomas) due to SV40 large T-antigen expression in pancreatic islet β cells. RABL6A loss in RT2 mice significantly delayed pancreatic tumor formation, reduced tumor angiogenesis and mitoses, and extended survival. Those effects correlated with upregulation of anti-angiogenic p19ARF and downregulation of proangiogenic in RABL6A-deficient islets and tumors. Our findings demonstrate that RABL6A is a bona fide oncogenic driver of pNET angiogenesis and development in vivo.
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http://dx.doi.org/10.3390/biomedicines9060633DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8228095PMC
June 2021

Development and comparison of novel bioluminescent mouse models of pancreatic neuroendocrine neoplasm metastasis.

Sci Rep 2021 05 13;11(1):10252. Epub 2021 May 13.

Department of Neuroscience and Pharmacology, University of Iowa, 2-570 Bowen Science Building, 51 Newton Road, Iowa City, IA, 52242, USA.

Pancreatic neuroendocrine neoplasms (pNENs) are slow growing cancers of increasing incidence that lack effective treatments once they become metastatic. Unfortunately, nearly half of pNEN patients present with metastatic liver tumors at diagnosis and current therapies fail to improve overall survival. Pre-clinical models of pNEN metastasis are needed to advance our understanding of the mechanisms driving the metastatic process and for the development of novel, targeted therapeutic interventions. To model metastatic dissemination of tumor cells, human pNEN cell lines (BON1 and Qgp1) stably expressing firefly luciferase (luc) were generated and introduced into NSG immunodeficient mice by intracardiac (IC) or intravenous (IV) injection. The efficiency, kinetics and distribution of tumor growth was evaluated weekly by non-invasive bioluminescent imaging (BLI). Tumors formed in all animals in both the IC and IV models. Bioluminescent Qgp1.luc cells preferentially metastasized to the liver regardless of delivery route, mimicking the predominant site of pNEN metastasis in patients. By comparison, BON1.luc cells most commonly formed lung tumors following either IV or IC administration and colonized a wider variety of tissues than Qgp1.luc cells. These models provide a unique platform for testing candidate metastasis genes and anti-metastatic therapies for pNENs.
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http://dx.doi.org/10.1038/s41598-021-89866-1DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8119958PMC
May 2021

Presacral neuroendocrine tumors associated with the Currarino syndrome.

Am J Med Genet A 2021 05 1;185(5):1582-1588. Epub 2021 Mar 1.

Department of Surgery, University of Iowa Carver College of Medicine, Iowa City, Iowa, USA.

Currarino syndrome (CS) is an autosomal dominant syndrome caused by mutations in MNX1 and characterized by anorectal abnormalities, partial sacral agenesis, and presacral masses. The presacral masses are typically benign; however, malignant degeneration can occur, and presacral neuroendocrine tumors (NETs) have been reported in six cases. We report three individuals from two families affected by CS in which multiple individuals developed presacral NETs. The first family, 491, had six members with features of CS, including two siblings who presented with presacral, Grade 2 NETs, one of which had metastasized to bone and lymph nodes. A germline c.874C>T (p.Arg292Trp) mutation was found in a highly conserved region of MNX1 in three affected members who underwent sequencing. A second somatic variant/deletion in MNX1 was not detected in either patient's tumor. In the second family, 342, the proband presented with an incidentally discovered presacral NET. The proband's father had previously undergone resection of a presacral NET, and so genetic testing was performed, which did not reveal an MNX1 mutation or copy number variants. The lack of a second, somatic mutation in the tumors from family 491 argues against MNX1 acting as a tumor suppressor, and the absence of a germline MNX1 mutation in family 342 suggests that other genetic and anatomic factors contribute to the development of presacral NETs. These cases highlight the variable presentation of CS, and the potential for malignancy in these patients.
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http://dx.doi.org/10.1002/ajmg.a.62145DOI Listing
May 2021

Combination therapies for MPNSTs targeting RABL6A-RB1 signaling.

Oncotarget 2021 Jan 5;12(1):10-14. Epub 2021 Jan 5.

Molecular Medicine Graduate Program, University of Iowa, Iowa City, Iowa, USA.

Precision medicine relies on a detailed molecular understanding of disease pathogenesis. Here, we consider urgently needed therapeutic options for malignant peripheral nerve sheath tumors (MPNSTs) based on emerging insights into druggable pathway alterations found to drive this deadly cancer. Recent observations demonstrate an essential role for an oncogenic GTPase, RABL6A, in promoting MPNST progression through hyperactivation of cyclin-dependent kinases (CDKs) and inactivation of the retinoblastoma (RB1) tumor suppressor. Monotherapies with CDK4/6 inhibitors have shown limited efficacy and durability in pre-clinical studies of MPNSTs and in clinical studies of other tumors. Therefore, we discuss the rationale and clinical benefits of inhibiting multiple RABL6A effectors, particularly CDK4/6 and MEK kinases, in targeted combination therapies suitable for MPNSTs and other Ras-driven malignancies.
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http://dx.doi.org/10.18632/oncotarget.27862DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7800773PMC
January 2021

Utility of Flow Cytometry and Fluorescence In Situ Hybridization in Follow-up Monitoring of Plasma Cell Myeloma.

Am J Clin Pathol 2021 07;156(2):198-204

Department of Pathology.

Objectives: We sought to investigate the clinical utility of flow cytometry (FC) and fluorescence in situ hybridization (FISH) in the workup of myeloma.

Methods: We retrospectively reviewed the reports of bone marrow biopsies received for myeloma evaluation between October 2015 and January 2019.

Results: A total of 1,708 biopsy specimens from 469 myeloma patients (mean age, 64.5 years [SD, 9.3]; female, 41.4%) were reviewed. Both FC and FISH had comparable detection rates at the time of initial diagnosis (97.6% vs 98.8%) and for follow-up cases (28.6% vs 28.2%). FC and FISH results were concordant in 98.8% of the initial diagnosis cases and 89.6% of the follow-up cases. The FISH-positive (FISH+)/FC-negative (FC-) discordance and FISH-/FC+ discordance occurred among 81 (5.0%) and 87 (5.4%) follow-up cases. In comparison with all concordant cases, FISH+/FC- discordant cases were more likely to have received treatment with daratumumab (P < .05).

Conclusions: Plasma cell-enriched FISH and FC have comparable abnormal plasma cell detection rates, and approximately 10% of the follow-up cases have discordant FISH and FC results in which residual disease is detected by only one of these modalities. FISH testing should be considered for cases with negative FC, especially in patients who have received treatment with daratumumab or in cases in which there is concern about specimen adequacy.
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http://dx.doi.org/10.1093/ajcp/aqaa224DOI Listing
July 2021

Using an aquatic model, Xenopus laevis, to uncover the role of chromodomain 1 in craniofacial disorders.

Genesis 2021 02 11;59(1-2):e23394. Epub 2020 Sep 11.

Department of Biology, Virginia Commonwealth University, Richmond, Virginia, USA.

The chromodomain family member chromodomain 1 (CHD1) has been shown to have numerous critical molecular functions including transcriptional regulation, splicing, and DNA repair. Complete loss of function of this gene is not compatible with life. On the other hand, missense and copy number variants of CHD1 can result in intellectual disabilities and craniofacial malformations in human patients including cleft palate and Pilarowski-Bjornsson Syndrome. We have used the aquatic developmental model organism Xenopus laevis, to determine a specific role for Chd1 in such cranioafcial disorders. Protein and gene knockdown techniques in Xenopus, including antisense oligos and mosaic Crispr/Cas9-mediated mutagenesis, recapitulated the craniofacial defects observed in humans. Further analysis indicated that embryos deficient in Chd1 had defects in cranial neural crest development and jaw cartilage morphology. Additionally, flow cytometry and immunohistochemistry revealed that decreased Chd1 resulted in increased in apoptosis in the developing head. Together, these experiments demonstrate that Chd1 is critical for fundamental processes and cell survival in craniofacial development. We also presented evidence that Chd1 is regulated by retinoic acid signaling during craniofacial development. Expression levels of chd1 mRNA, specifically in the head, were increased by RAR agonist exposure and decreased upon antagonist treatment. Subphenotypic levels of an RAR antagonist and Chd1 morpholinos synergized to result in orofacial defects. Further, RAR DNA binding sequences (RAREs) were detected in chd1 regulatory regions by bioinformatic analysis. In summary, by combining human genetics and experiments in an aquatic model we now have a better understanding of the role of CHD1 in craniofacial disorders.
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http://dx.doi.org/10.1002/dvg.23394DOI Listing
February 2021

Progressive optic disc cupping over 20 years in a patient with -associated glaucoma.

Ophthalmol Glaucoma 2020 Mar-Apr;3(2):167-168. Epub 2019 Nov 16.

Department of Ophthalmology and Visual Sciences, Carver College of Medicine, University of Iowa, Iowa City, IA USA.

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http://dx.doi.org/10.1016/j.ogla.2019.11.003DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7337276PMC
August 2021

Expanding the genotypic and phenotypic spectrum of severe serine biosynthesis disorders.

Hum Mutat 2020 09 15;41(9):1615-1628. Epub 2020 Jul 15.

Institute of Human Genetics, University Hospital Magdeburg, Magdeburg, Germany.

Serine biosynthesis disorders comprise a spectrum of very rare autosomal recessive inborn errors of metabolism with wide phenotypic variability. Neu-Laxova syndrome represents the most severe expression and is characterized by multiple congenital anomalies and pre- or perinatal lethality. Here, we present the mutation spectrum and a detailed phenotypic analysis in 15 unrelated families with severe types of serine biosynthesis disorders. We identified likely disease-causing variants in the PHGDH and PSAT1 genes, several of which have not been reported previously. Phenotype analysis and a comprehensive review of the literature corroborates the evidence that serine biosynthesis disorders represent a continuum with varying degrees of phenotypic expression and suggest that even gradual differences at the severe end of the spectrum may be correlated with particular genotypes. We postulate that the individual residual enzyme activity of mutant proteins is the major determinant of the phenotypic variability, but further functional studies are needed to explore effects at the enzyme protein level.
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http://dx.doi.org/10.1002/humu.24067DOI Listing
September 2020

A missense mutation in the MLKL brace region promotes lethal neonatal inflammation and hematopoietic dysfunction.

Nat Commun 2020 06 19;11(1):3150. Epub 2020 Jun 19.

The Walter and Eliza Hall Institute of Medical Research, Parkville, VIC, 3052, Australia.

MLKL is the essential effector of necroptosis, a form of programmed lytic cell death. We have isolated a mouse strain with a single missense mutation, Mlkl, that alters the two-helix 'brace' that connects the killer four-helix bundle and regulatory pseudokinase domains. This confers constitutive, RIPK3 independent killing activity to MLKL. Homozygous mutant mice develop lethal postnatal inflammation of the salivary glands and mediastinum. The normal embryonic development of Mlkl homozygotes until birth, and the absence of any overt phenotype in heterozygotes provides important in vivo precedent for the capacity of cells to clear activated MLKL. These observations offer an important insight into the potential disease-modulating roles of three common human MLKL polymorphisms that encode amino acid substitutions within or adjacent to the brace region. Compound heterozygosity of these variants is found at up to 12-fold the expected frequency in patients that suffer from a pediatric autoinflammatory disease, chronic recurrent multifocal osteomyelitis (CRMO).
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http://dx.doi.org/10.1038/s41467-020-16819-zDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7305203PMC
June 2020

Longitudinal phenotype development in a minipig model of neurofibromatosis type 1.

Sci Rep 2020 03 19;10(1):5046. Epub 2020 Mar 19.

Department of Radiology, University of Iowa, Iowa City, IA, USA.

Neurofibromatosis type 1 (NF1) is a rare, autosomal dominant disease with variable clinical presentations. Large animal models are useful to help dissect molecular mechanisms, determine relevant biomarkers, and develop effective therapeutics. Here, we studied a NF1 minipig model (NF1) for the first 12 months of life to evaluate phenotype development, track disease progression, and provide a comparison to human subjects. Through systematic evaluation, we have shown that compared to littermate controls, the NF1 model develops phenotypic characteristics of human NF1: [1] café-au-lait macules, [2] axillary/inguinal freckling, [3] shortened stature, [4] tibial bone curvature, and [5] neurofibroma. At 4 months, full body computed tomography imaging detected significantly smaller long bones in NF1 minipigs compared to controls, indicative of shorter stature. We found quantitative evidence of tibial bowing in a subpopulation of NF1 minipigs. By 8 months, an NF1 boar developed a large diffuse shoulder neurofibroma, visualized on magnetic resonance imaging, which subsequently grew in size and depth as the animal aged up to 20 months. The NF1 minipig model progressively demonstrates signature attributes that parallel clinical manifestations seen in humans and provides a viable tool for future translational NF1 research.
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http://dx.doi.org/10.1038/s41598-020-61251-4DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7081358PMC
March 2020

RABL6A Is an Essential Driver of MPNSTs that Negatively Regulates the RB1 Pathway and Sensitizes Tumor Cells to CDK4/6 Inhibitors.

Clin Cancer Res 2020 06 21;26(12):2997-3011. Epub 2020 Feb 21.

Molecular Medicine Graduate Program, University of Iowa, Iowa City, Iowa.

Purpose: Malignant peripheral nerve sheath tumors (MPNST) are deadly sarcomas that lack effective therapies. In most MPNSTs, the retinoblastoma (RB1) tumor suppressor is disabled by hyperactivation of cyclin-dependent kinases (CDK), commonly through loss of CDK-inhibitory proteins such as p27(Kip1). RABL6A is an inhibitor of RB1 whose role in MPNSTs is unknown. To gain insight into MPNST development and establish new treatment options, we investigated RABL6A-RB1 signaling and CDK inhibitor-based therapy in MPNSTs.

Experimental Design: We examined patient-matched MPNSTs and precursor lesions by RNA sequencing (RNA-Seq) and IHC. Molecular and biological effects of silencing RABL6A and/or p27 in MPNST lines and normal human Schwann cells were determined. Tumor-suppressive effects of CDK inhibitors were measured in MPNST cells and orthotopic tumors.

Results: RABL6A was dramatically upregulated in human MPNSTs compared with precursor lesions, which correlated inversely with p27 levels. Silencing RABL6A caused MPNST cell death and G arrest that coincided with p27 upregulation, CDK downregulation, and RB1 activation. The growth-suppressive effects of RABL6A loss, and its regulation of RB1, were largely rescued by p27 depletion. Importantly, reactivation of RB1 using a CDK4/6 inhibitor (palbociclib) killed MPNST cells in an RABL6A-dependent manner and suppressed MPNST growth . Low-dose combination of drugs targeting multiple RB1 kinases (CDK4/6, CDK2) had enhanced antitumorigenic activity associated with potential MPNST cell redifferentiation.

Conclusions: RABL6A is a new driver of MPNST pathogenesis that acts in part through p27-RB1 inactivation. Our results suggest RB1 targeted therapy with multiple pathway drugs may effectively treat MPNSTs.
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http://dx.doi.org/10.1158/1078-0432.CCR-19-2706DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7299809PMC
June 2020

Interstitial duplication of 8q22.1-q23.1- A case report and review of the literature.

Clin Case Rep 2019 Dec 24;7(12):2393-2397. Epub 2019 Oct 24.

Division of Medical Genetics and Genomics The Stead Family Department of Pediatrics University of Iowa Hospitals and Clinics Iowa City IA USA.

This report highlights the clinical features seen in duplication of 8q22.1q23.1 inherited from balanced father. It stresses the importance of obtaining a karyotype to identify the location of a large copy number variant for accurate recurrence risk estimation.
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http://dx.doi.org/10.1002/ccr3.2507DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6935641PMC
December 2019

Candidate modifier genes for immune function in 22q11.2 deletion syndrome.

Mol Genet Genomic Med 2020 01 12;8(1):e1057. Epub 2019 Dec 12.

Stead Family Department of Pediatrics, Iowa City, IA, USA.

Background: The 22q11.2 deletion syndrome (22q11.2DS) is the most common contiguous microdeletion affecting humans and exhibits extreme phenotypic heterogeneity. Patients can manifest any combination of comorbidities including congenital heart disease, hypoparathyroidism, cleft palate, kidney abnormalities, neurodevelopmental disorders, and immune dysfunction. Immunodeficiency is present in the majority of patients with 22q11.2DS and is the second leading cause of death in these patients. Knowing the genetic determinants of immune dysfunction will aid in prognostication and potentially novel treatments.

Methods: We performed exome sequencing and gene-based variant association analysis on 31 deeply phenotyped individuals with the canonical 3Mb 22q11.2 deletion to identify what genes outside the 22q11.2 locus may be modifying the immune dysregulated phenotype. Immunophenotyping was performed using preexisting medical data and a novel scoring system developed from numerous clinical laboratory values including immunoglobulin levels, lymphocyte transformation to antigens (LTA), lymphocyte transformation to mitogens (LTM), and peripheral blood flow cytometry. Immunophenotypic scoring was validated against newborn screening T-cell receptor excision circle (TREC) results.

Results: Rare DNA variants in transcriptional regulators involved in retinoic acid signaling (NCOR2, OMIM *600848 and EP300, OMIM *602700) were found to be associated with immunophenotype.

Conclusion: The expression of TBX1, which seems to confer the major phenotypic features of 22q11.2DS, is regulated via retinoic acid signaling, and alterations in retinoic acid signaling during embryonic development can lead to phenocopies of 22q11.2DS. These observations support the hypothesis that genetic modifiers outside the microdeletion locus may influence the immune function in 22q11.2DS patients.
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http://dx.doi.org/10.1002/mgg3.1057DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6978229PMC
January 2020

Novel Intragenic Deletion in a Pedigree with Aniridia, Morbid Obesity, and Diabetes.

Curr Eye Res 2020 01 14;45(1):91-96. Epub 2019 Aug 14.

Department of Ophthalmology and Visual Sciences, Carver College of Medicine, University of Iowa, Iowa City, IA, USA.

: Aniridia is a rare congenital eye disease, characterized by a constellation of symptoms including hypoplastic irides, foveal hypoplasia, early cataract, corneal stem cell deficiency, and glaucoma. Large chromosomal deletions spanning the gene cause WAGR syndrome (Wilms tumor, aniridia, genitourinary anomalies, and intellectual disability [formerly called mental retardation]). We describe clinical and genetic studies of a three-generation pedigree with aniridia along with additional systemic conditions (morbid obesity, diabetes) suggesting the possibility of a contiguous-gene syndrome like WAGR.: Clinical records were obtained and DNA was prepared from blood samples from three of the four patients and tested for mutations in the coding sequences of the gene. The index patient also had cardiomyopathy and was tested for known cardiomyopathy genetic mutations using a next-generation DNA sequencing assay.: We discovered a novel intragenic mutation, a 16 bp heterozygous deletion c.203delCCAGGGCAATCGGTGG, with Sanger sequencing that is the likely cause of autosomal dominant aniridia in this pedigree. This deletion causes a frameshift in predicted protein translation and a subsequent premature termination, p.Pro68Leufs*6. The deletion was detected in all three available family members with aniridia, the index patient, his mother, and his maternal aunt but was not observed in the ome ggregation onsortium (ExAC) database. Targeted sequencing of known cardiomyopathy genes in the index patient identified a second mutation, a 1.7 Mp deletion that spans the gene.: We report a pedigree with aniridia and other systemic abnormalities that were initially suspicious for a contiguous-gene syndrome like WAGR. However, genetic analysis of the pedigree revealed two independent genetic abnormalities on chromosome 11p: 1) a novel mutation, and 2) a large chromosome deletion spanning , a known cardiomyopathy gene. It is unclear if morbid obesity and type II diabetes mellitus have a related genetic cause.
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http://dx.doi.org/10.1080/02713683.2019.1649704DOI Listing
January 2020

Assessment of nociception and related quality-of-life measures in a porcine model of neurofibromatosis type 1.

Pain 2019 11;160(11):2473-2486

Pediatrics and Rare Diseases Group, Sanford Research, Sioux Falls, SD, United States.

Neurofibromatosis type 1 (NF1) is an autosomal dominant genetic disorder resulting from germline mutations in the NF1 gene, which encodes neurofibromin. Patients experience a variety of symptoms, but pain in the context of NF1 remains largely underrecognized. Here, we characterize nociceptive signaling and pain behaviors in a miniswine harboring a disruptive NF1 mutation (exon 42 deletion). We present the first characterization of pain-related behaviors in a pig model of NF1, identifying unchanged agitation scores, lower tactile thresholds (allodynia), and decreased response latencies to thermal laser stimulation (hyperalgesia) in NF1 (females only) pigs. Male NF1 pigs with tumors showed reduced sleep quality and increased resting, 2 health-related quality-of-life symptoms found to be comorbid in people with NF1 pain. We explore these phenotypes in relationship to suppression of the increased activity of the N-type voltage-gated calcium (CaV2.2) channel by pharmacological antagonism of phosphorylation of a regulatory protein-the collapsin response mediator protein 2 (CRMP2), a known interactor of neurofibromin, and by targeting the interface between the α subunit of CaV2.2 and the accessory β-subunits with small molecules. Our data support the use of NF1 pigs as a large animal model for studying NF1-associated pain and for understanding the pathophysiology of NF1. Our findings demonstrate the translational potential of 2 small molecules in reversing ion channel remodeling seen in NF1. Interfering with CaV2.2, a clinically validated target for pain management, might also be a promising therapeutic strategy for NF1-related pain management.
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http://dx.doi.org/10.1097/j.pain.0000000000001648DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6800626PMC
November 2019

In trans variant calling reveals enrichment for compound heterozygous variants in genes involved in neuronal development and growth.

Genet Res (Camb) 2019 06 13;101:e8. Epub 2019 Jun 13.

Department of Pediatrics,The University of Iowa,Iowa City,IA,USA.

Compound heterozygotes occur when different variants at the same locus on both maternal and paternal chromosomes produce a recessive trait. Here we present the tool VarCount for the quantification of variants at the individual level. We used VarCount to characterize compound heterozygous coding variants in patients with epileptic encephalopathy and in the 1000 Genomes Project participants. The Epi4k data contains variants identified by whole exome sequencing in patients with either Lennox-Gastaut Syndrome (LGS) or infantile spasms (IS), as well as their parents. We queried the Epi4k dataset (264 trios) and the phased 1000 Genomes Project data (2504 participants) for recessive variants. To assess enrichment, transcript counts were compared between the Epi4k and 1000 Genomes Project participants using minor allele frequency (MAF) cutoffs of 0.5 and 1.0%, and including all ancestries or only probands of European ancestry. In the Epi4k participants, we found enrichment for rare, compound heterozygous variants in six genes, including three involved in neuronal growth and development - PRTG (p = 0.00086, 1% MAF, combined ancestries), TNC (p = 0.022, 1% MAF, combined ancestries) and MACF1 (p = 0.0245, 0.5% MAF, EU ancestry). Due to the total number of transcripts considered in these analyses, the enrichment detected was not significant after correction for multiple testing and higher powered or prospective studies are necessary to validate the candidacy of these genes. However, PRTG, TNC and MACF1 are potential novel recessive epilepsy genes and our results highlight that compound heterozygous variants should be considered in sporadic epilepsy.
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http://dx.doi.org/10.1017/S0016672319000065DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7045018PMC
June 2019

RABL6A inhibits tumor-suppressive PP2A/AKT signaling to drive pancreatic neuroendocrine tumor growth.

J Clin Invest 2019 03 4;129(4):1641-1653. Epub 2019 Mar 4.

Department of Pharmacology.

Hyperactivated AKT/mTOR signaling is a hallmark of pancreatic neuroendocrine tumors (PNETs). Drugs targeting this pathway are used clinically, but tumor resistance invariably develops. A better understanding of factors regulating AKT/mTOR signaling and PNET pathogenesis is needed to improve current therapies. We discovered that RABL6A, a new oncogenic driver of PNET proliferation, is required for AKT activity. Silencing RABL6A caused PNET cell-cycle arrest that coincided with selective loss of AKT-S473 (not T308) phosphorylation and AKT/mTOR inactivation. Restoration of AKT phosphorylation rescued the G1 phase block triggered by RABL6A silencing. Mechanistically, loss of AKT-S473 phosphorylation in RABL6A-depleted cells was the result of increased protein phosphatase 2A (PP2A) activity. Inhibition of PP2A restored phosphorylation of AKT-S473 in RABL6A-depleted cells, whereas PP2A reactivation using a specific small-molecule activator of PP2A (SMAP) abolished that phosphorylation. Moreover, SMAP treatment effectively killed PNET cells in a RABL6A-dependent manner and suppressed PNET growth in vivo. The present work identifies RABL6A as a new inhibitor of the PP2A tumor suppressor and an essential activator of AKT in PNET cells. Our findings offer what we believe is a novel strategy of PP2A reactivation for treatment of PNETs as well as other human cancers driven by RABL6A overexpression and PP2A inactivation.
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http://dx.doi.org/10.1172/JCI123049DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6436899PMC
March 2019

A case of primary cutaneous Ewing sarcoma in a neutropenic patient.

J Cutan Pathol 2019 Mar 16;46(3):238-241. Epub 2019 Jan 16.

Department of Dermatology, University of Iowa Hospitals and Clinics, Iowa City, Iowa.

Primary cutaneous Ewing sarcoma is a rare clinical presentation of Ewing sarcoma, usually occurring as a small, localized tumor on the extremities of young adults and associated with favorable prognosis. We report a case of primary cutaneous Ewing sarcoma, which presented on the sole of the foot of a 27-year-old patient with relapsed acute myeloid leukemia and neutropenia. Diagnosis was determined through histological features and staining, as well as fluorescence in situ hybridization and molecular testing. The patient underwent wide-local excision with plan to begin targeted chemotherapy, but unfortunately died from adenovirus pneumonia while neutropenic before targeted chemotherapy was initiated.
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http://dx.doi.org/10.1111/cup.13406DOI Listing
March 2019

A porcine model of neurofibromatosis type 1 that mimics the human disease.

JCI Insight 2018 06 21;3(12). Epub 2018 Jun 21.

Pediatrics and Rare Diseases Group, Sanford Research, Sioux Falls, South Dakota, USA.

Loss of the NF1 tumor suppressor gene causes the autosomal dominant condition, neurofibromatosis type 1 (NF1). Children and adults with NF1 suffer from pathologies including benign and malignant tumors to cognitive deficits, seizures, growth abnormalities, and peripheral neuropathies. NF1 encodes neurofibromin, a Ras-GTPase activating protein, and NF1 mutations result in hyperactivated Ras signaling in patients. Existing NF1 mutant mice mimic individual aspects of NF1, but none comprehensively models the disease. We describe a potentially novel Yucatan miniswine model bearing a heterozygotic mutation in NF1 (exon 42 deletion) orthologous to a mutation found in NF1 patients. NF1+/ex42del miniswine phenocopy the wide range of manifestations seen in NF1 patients, including café au lait spots, neurofibromas, axillary freckling, and neurological defects in learning and memory. Molecular analyses verified reduced neurofibromin expression in swine NF1+/ex42del fibroblasts, as well as hyperactivation of Ras, as measured by increased expression of its downstream effectors, phosphorylated ERK1/2, SIAH, and the checkpoint regulators p53 and p21. Consistent with altered pain signaling in NF1, dysregulation of calcium and sodium channels was observed in dorsal root ganglia expressing mutant NF1. Thus, these NF1+/ex42del miniswine recapitulate the disease and provide a unique, much-needed tool to advance the study and treatment of NF1.
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http://dx.doi.org/10.1172/jci.insight.120402DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6124439PMC
June 2018

Uniparental disomy unveils a novel recessive mutation in POMT2.

Neuromuscul Disord 2018 07 10;28(7):592-596. Epub 2018 Apr 10.

Department of Pediatrics, Roy J. and Lucille A. Carver College of Medicine, University of Iowa, Iowa City, Iowa, USA; Department of Neurology, Roy J. and Lucille A. Carver College of Medicine, University of Iowa, Iowa City, Iowa, USA. Electronic address:

Mutations in POMT2 are most commonly associated with Walker-Warburg syndrome and Muscle-Eye-Brain disease, but can also cause limb girdle muscular dystrophy (LGMD2N). We report a case of LGMD due to a novel mutation in POMT2 unmasked by uniparental isodisomy. The patient experienced proximal muscle weakness from three years of age with minimal progression. She developed progressive contractures and underwent unilateral Achilles tenotomy. By age 11, she had borderline low left ventricular ejection fraction and mild restrictive lung disease. Muscle biopsy showed mild dystrophic changes with selective reduction in α-dystroglycan immunostaining. Sequencing of POMT2 showed a novel homozygous c.1502A>C variant that was predicted to be probably pathogenic. Fibroblast complementation studies showed lack of functional glycosylation rescued by wild-type POMT2 expression. Chromosomal microarray showed a single 15 Mb copy number neutral loss of heterozygosity on chromosome 14 encompassing POMT2. RNAseq verified homozygosity at this locus. Together, our findings indicate maternal uniparental isodisomy causing LGMD2N.
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http://dx.doi.org/10.1016/j.nmd.2018.04.003DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6115279PMC
July 2018

Identification of as a Novel Clefting and Craniofacial Patterning Gene in Humans.

Genetics 2018 01 21;208(1):283-296. Epub 2017 Nov 21.

Department of Pediatrics,

Orofacial clefts are one of the most common birth defects, affecting 1-2 per 1000 births, and have a complex etiology. High-resolution array-based comparative genomic hybridization has increased the ability to detect copy number variants (CNVs) that can be causative for complex diseases such as cleft lip and/or palate. Utilizing this technique on 97 nonsyndromic cleft lip and palate cases and 43 cases with cleft palate only, we identified a heterozygous deletion of in one affected case, as well as a deletion in a second case that removes putative 3' regulatory information. is a strong candidate for clefting, as it is expressed in orofacial structures derived from the first branchial arch and is also in the same "synexpression group" as and and , all of which have been associated with clefting. CNVs affecting are exceedingly rare in control populations, and scores as a likely haploinsufficiency locus. Confirming its role in craniofacial development, knockdown or clustered randomly interspaced short palindromic repeats/Cas9-generated mutation of in resulted in mild to severe craniofacial dysmorphologies, with several individuals presenting with median clefts. Moreover, knockdown of produced decreased expression of , itself a gene associated with clefting, in regions of the face that pattern the maxilla. Our study demonstrates a successful pipeline from CNV identification of a candidate gene to functional validation in a vertebrate model system, and reveals as both a new human clefting locus as well as a key craniofacial patterning gene.
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http://dx.doi.org/10.1534/genetics.117.300535DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5753863PMC
January 2018

Immunohistochemical Markers for Prospective Studies in Neurofibromatosis-1 Porcine Models.

J Histochem Cytochem 2017 10 28;65(10):607-618. Epub 2017 Aug 28.

Pediatrics and Rare Disease Group, Sanford Research, Sioux Falls, South Dakota.

Neurofibromatosis type 1 (NF1) is a common, cancer-predisposing disease caused by mutations in the NF1 tumor gene. Patients with NF1 have an increased risk for benign and malignant tumors of the nervous system (e.g., neurofibromas, malignant peripheral nerve sheath tumors, gliomas) and other tissues (e.g., leukemias, rhabdomyosarcoma, etc.) as well as increased susceptibility to learning disabilities, chronic pain/migraines, hypertension, pigmentary changes, and developmental lesions (e.g., tibial pseudoarthrosis). Pigs are an attractive and upcoming animal model for future NF1 studies, but a potential limitation to porcine model research has been the lack of validated reagents for direct translational study to humans. To address that issue, we used formalin-fixed tissues (human and pigs) to evaluate select immunohistochemical markers (activated caspase-3, allograft inflammatory factor-1, beta-tubulin III, calbindin D, CD13, CD20, desmin, epithelial membrane antigen, glial fibrillary acidic protein, glucose transporter-1, laminin, myelin basic protein, myoglobin, proliferating cell nuclear antigen, S100, vimentin, and von Willebrand factor). The markers were validated by comparing known expression and localization in human and pig tissues. Validation of these markers on fixed tissues will facilitate prospective immunohistochemical studies of NF1 pigs, as well as other pig models, in a more efficient, reproducible, and translationally relevant manner.
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http://dx.doi.org/10.1369/0022155417729357DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5624366PMC
October 2017

Development of Secondary Acute Myeloid Leukemia in a Pediatric Patient Concurrently Receiving Primary Therapy for Ewing Sarcoma.

J Pediatr Hematol Oncol 2017 10;39(7):e370-e372

*University of Iowa Hospitals and Clinics, Iowa City, IA †Blank Children's Cancer and Blood Disorder Center, Des Moines, IA.

Ewing sarcoma is a pediatric bone and soft tissue sarcoma that requires intensive therapy, which can cause secondary malignancies. We present a rare case of early, treatment-related AML in a pediatric patient concurrently receiving primary therapy for Ewing sarcoma. Despite AML-directed therapy, our patient died secondary to complications of hyperleukocytosis. Cytogenetic and mutation profiling of the leukemia cells revealed the DNA-topoisomerase-II-inhibitor-associated t(9;11)(p22;q23) translocation and clonal KRAS and BRAF mutations. This report highlights the importance of monitoring for treatment-related effects in cancer therapy, as well as the need for novel, less toxic approaches in Ewing sarcoma therapy.
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http://dx.doi.org/10.1097/MPH.0000000000000924DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5772896PMC
October 2017

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

Genetics 2017 09 24;207(1):215-228. Epub 2017 Jul 24.

Department of Pediatrics, University of Iowa, Iowa City, Iowa 52242.

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 , , and 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, Analysis of a zebrafish loss-of-function mutant revealed defects in the pronephric kidney just prior to death, and F0 CRISPR/Cas9 mutagenesis of in the mouse revealed kidney agenesis phenotypes, implicating 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.
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http://dx.doi.org/10.1534/genetics.117.1125DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5586373PMC
September 2017

Recessive coding and regulatory mutations in FBLIM1 underlie the pathogenesis of chronic recurrent multifocal osteomyelitis (CRMO).

PLoS One 2017 16;12(3):e0169687. Epub 2017 Mar 16.

Department of Pediatrics, The University of Iowa, Iowa City, IA, United States of America.

Chronic recurrent multifocal osteomyelitis (CRMO) is a rare, pediatric, autoinflammatory disease characterized by bone pain due to sterile osteomyelitis, and is often accompanied by psoriasis or inflammatory bowel disease. There are two syndromic forms of CRMO, Majeed syndrome and DIRA, for which the genetic cause is known. However, for the majority of cases of CRMO, the genetic basis is unknown. Via whole-exome sequencing, we detected a homozygous mutation in the filamin-binding domain of FBLIM1 in an affected child with consanguineous parents. Microarray analysis of bone marrow macrophages from the CRMO murine model (cmo) determined that the Fblim1 ortholog is the most differentially expressed gene, downregulated over 20-fold in the cmo mouse. We sequenced FBLIM1 in 96 CRMO subjects and found a second proband with a novel frameshift mutation in exon 6 and a rare regulatory variant. In SaOS2 cells, overexpressing the regulatory mutation showed the flanking region acts as an enhancer, and the mutation ablates enhancer activity. Our data implicate FBLIM1 in the pathogenesis of sterile bone inflammation and our findings suggest CRMO is a disorder of chronic inflammation and imbalanced bone remodeling.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0169687PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5354242PMC
September 2017

The Eating-Disorder Associated HDAC4 Mutation Alters Feeding Behaviors in Female Mice.

Biol Psychiatry 2017 05 13;81(9):770-777. Epub 2016 Oct 13.

Pharmacology, University of Iowa, Carver College of Medicine, Iowa City, Iowa. Electronic address:

Background: While eating disorders (EDs) are thought to result from a combination of environmental and psychological stressors superimposed on genetic vulnerability, the neurobiological basis of EDs remains incompletely understood. We recently reported that a rare missense mutation in the gene for the transcriptional repressor histone deacetylase 4 (HDAC4) is associated with the risk of developing an ED in humans.

Methods: To understand the biological consequences of this missense mutation, we created transgenic mice carrying this mutation by introducing the alanine to threonine mutation at position 778 of mouse Hdac4 (corresponding to position 786 of the human protein). Bioinformatic analysis to identify Hdac4-regulated genes was performed using available databases.

Results: Male mice heterozygous for HDAC4 did not show any metabolic or behavioral differences. In contrast, female mice heterozygous for HDAC4 display several ED-related feeding and behavioral deficits depending on housing condition. Individually housed HDAC4 female mice exhibit reduced effortful responding for high-fat diet and compulsive grooming, whereas group-housed female mice display increased weight gain on high-fat diet, reduced behavioral despair, and increased anxiety-like behaviors. Bioinformatic analysis identifies mitochondrial biogenesis including synthesis of glutamate/gamma-aminobutyric acid as a potential transcriptional target of HDAC4 activity relevant to the behavioral deficits identified in this new mouse model of disordered eating.

Conclusions: The HDAC4 mouse line is a novel model of ED-related behaviors and identifies mitochondrial biogenesis as a potential molecular pathway contributing to behavioral deficits.
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http://dx.doi.org/10.1016/j.biopsych.2016.09.024DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5386818PMC
May 2017

Mutations of the Sonic Hedgehog Pathway Underlie Hypothalamic Hamartoma with Gelastic Epilepsy.

Am J Hum Genet 2016 08 21;99(2):423-9. Epub 2016 Jul 21.

Institute for Genomic Medicine, Columbia University, New York, NY 10032, USA; Department of Pathology and Cell Biology, Columbia University, New York, NY 10032, USA. Electronic address:

Hypothalamic hamartoma (HH) with gelastic epilepsy is a well-recognized drug-resistant epilepsy syndrome of early life.(1) Surgical resection allows limited access to the small deep-seated lesions that cause the disease. Here, we report the results of a search for somatic mutations in paired hamartoma- and leukocyte-derived DNA samples from 38 individuals which we conducted by using whole-exome sequencing (WES), chromosomal microarray (CMA), and targeted resequencing (TRS) of candidate genes. Somatic mutations were identified in genes involving regulation of the sonic hedgehog (Shh) pathway in 14/38 individuals (37%). Three individuals had somatic mutations in PRKACA, which encodes a cAMP-dependent protein kinase that acts as a repressor protein in the Shh pathway, and four subjects had somatic mutations in GLI3, an Shh pathway gene associated with HH. In seven other individuals, we identified two recurrent and three single brain-tissue-specific, large copy-number or loss-of-heterozygosity (LOH) variants involving multiple Shh genes, as well as other genes without an obvious biological link to the Shh pathway. The Shh pathway genes in these large somatic lesions include the ligand itself (SHH and IHH), the receptor SMO, and several other Shh downstream pathway members, including CREBBP and GLI2. Taken together, our data implicate perturbation of the Shh pathway in at least 37% of individuals with the HH epilepsy syndrome, consistent with the concept of a developmental pathway brain disease.
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http://dx.doi.org/10.1016/j.ajhg.2016.05.031DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4974069PMC
August 2016

Autism Linked to Increased Oncogene Mutations but Decreased Cancer Rate.

PLoS One 2016 2;11(3):e0149041. Epub 2016 Mar 2.

Interdisciplinary Program in Genetics, University of Iowa, Iowa City, Iowa, United States of America.

Autism spectrum disorder (ASD) is one phenotypic aspect of many monogenic, hereditary cancer syndromes. Pleiotropic effects of cancer genes on the autism phenotype could lead to repurposing of oncology medications to treat this increasingly prevalent neurodevelopmental condition for which there is currently no treatment. To explore this hypothesis we sought to discover whether autistic patients more often have rare coding, single-nucleotide variants within tumor suppressor and oncogenes and whether autistic patients are more often diagnosed with neoplasms. Exome-sequencing data from the ARRA Autism Sequencing Collaboration was compared to that of a control cohort from the Exome Variant Server database revealing that rare, coding variants within oncogenes were enriched for in the ARRA ASD cohort (p<1.0 x 10(-8)). In contrast, variants were not significantly enriched in tumor suppressor genes. Phenotypically, children and adults with ASD exhibited a protective effect against cancer, with a frequency of 1.3% vs. 3.9% (p<0.001), but the protective effect decreased with age. The odds ratio of neoplasm for those with ASD relative to controls was 0.06 (95% CI: 0.02, 0.19; p<0.0001) in the 0 to 14 age group; 0.35 (95% CI: 0.14, 0.87; p = 0.024) in the 15 to 29 age group; 0.41 (95% CI: 0.15, 1.17; p = 0.095) in the 30 to 54 age group; and 0.49 (95% CI: 0.14, 1.74; p = 0.267) in those 55 and older. Both males and females demonstrated the protective effect. These findings suggest that defects in cellular proliferation, and potentially senescence, might influence both autism and neoplasm, and already approved drugs targeting oncogenic pathways might also have therapeutic value for treating autism.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0149041PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4774916PMC
July 2016
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