Publications by authors named "Jill A Madden"

23 Publications

  • Page 1 of 1

Belzutifan, a Potent HIF2α Inhibitor, in the Pacak-Zhuang Syndrome.

N Engl J Med 2021 11;385(22):2059-2065

From the Departments of Pediatric Oncology (J.K., K.V.H., J.A.P., C.M.C., A.I., C.B.W., K.A.J., S.G.D.) and Medical Oncology (W.G.K.), Dana-Farber Cancer Institute, Harvard Medical School, the Divisions of Hematology and Oncology (J.K., J.A.P., M.M.H., K.A.J., S.G.D.) and Endocrinology (A.J.W.) and the Departments of Surgery (B.R.W.), Pathology (S.O.V.), and Radiology (S.D.V.), Boston Children's Hospital, Harvard Medical School, and the Manton Center for Orphan Disease Research and the Division of Genetics and Genomics, Boston Children's Hospital (J.A.M., J.L.) - all in Boston; Howard Hughes Medical Institute, Chevy Chase, MD (W.G.K.); and Merck, Kenilworth, NJ (R.F.P., N.J.Z.).

The integration of genomic testing into clinical care enables the use of individualized approaches to the management of rare diseases. We describe the use of belzutifan, a potent and selective small-molecule inhibitor of the protein hypoxia-inducible factor 2α (HIF2α), in a patient with polycythemia and multiple paragangliomas (the Pacak-Zhuang syndrome). The syndrome was caused in this patient by somatic mosaicism for an activating mutation in . Treatment with belzutifan led to a rapid and sustained tumor response along with resolution of hypertension, headaches, and long-standing polycythemia. This case shows the application of a targeted therapy for the treatment of a patient with a rare tumor-predisposition syndrome. (Funded by the Morin Family Fund for Pediatric Cancer and Alex's Lemonade Stand Foundation.).
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http://dx.doi.org/10.1056/NEJMoa2110051DOI Listing
November 2021

Novel variants in KAT6B spectrum of disorders expand our knowledge of clinical manifestations and molecular mechanisms.

Mol Genet Genomic Med 2021 Oct 14;9(10):e1809. Epub 2021 Sep 14.

Division of Genetics and Genomics, Department of Pediatrics, Boston Children's Hospital, Harvard Medical School, Boston, Massachusetts, USA.

The phenotypic variability associated with pathogenic variants in Lysine Acetyltransferase 6B (KAT6B, a.k.a. MORF, MYST4) results in several interrelated syndromes including Say-Barber-Biesecker-Young-Simpson Syndrome and Genitopatellar Syndrome. Here we present 20 new cases representing 10 novel KAT6B variants. These patients exhibit a range of clinical phenotypes including intellectual disability, mobility and language difficulties, craniofacial dysmorphology, and skeletal anomalies. Given the range of features previously described for KAT6B-related syndromes, we have identified additional phenotypes including concern for keratoconus, sensitivity to light or noise, recurring infections, and fractures in greater numbers than previously reported. We surveyed clinicians to qualitatively assess the ways families engage with genetic counselors upon diagnosis. We found that 56% (10/18) of individuals receive diagnoses before the age of 2 years (median age = 1.96 years), making it challenging to address future complications with limited accessible information and vast phenotypic severity. We used CRISPR to introduce truncating variants into the KAT6B gene in model cell lines and performed chromatin accessibility and transcriptome sequencing to identify key dysregulated pathways. This study expands the clinical spectrum and addresses the challenges to management and genetic counseling for patients with KAT6B-related disorders.
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http://dx.doi.org/10.1002/mgg3.1809DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8580094PMC
October 2021

A data-driven architecture using natural language processing to improve phenotyping efficiency and accelerate genetic diagnoses of rare disorders.

HGG Adv 2021 Jul 11;2(3). Epub 2021 May 11.

The Manton Center for Orphan Disease Research, Division of Genetics and Genomics, Boston Children's Hospital, Harvard Medical School, Boston, MA 02115, USA.

Effective genetic diagnosis requires the correlation of genetic variant data with detailed phenotypic information. However, manual encoding of clinical data into machine-readable forms is laborious and subject to observer bias. Natural language processing (NLP) of electronic health records has great potential to enhance reproducibility at scale but suffers from idiosyncrasies in physician notes and other medical records. We developed methods to optimize NLP outputs for automated diagnosis. We filtered NLP-extracted Human Phenotype Ontology (HPO) terms to more closely resemble manually extracted terms and identified filter parameters across a three-dimensional space for optimal gene prioritization. We then developed a tiered pipeline that reduces manual effort by prioritizing smaller subsets of genes to consider for genetic diagnosis. Our filtering pipeline enabled NLP-based extraction of HPO terms to serve as a sufficient replacement for manual extraction in 92% of prospectively evaluated cases. In 75% of cases, the correct causal gene was ranked higher with our applied filters than without any filters. We describe a framework that can maximize the utility of NLP-based phenotype extraction for gene prioritization and diagnosis. The framework is implemented within a cloud-based modular architecture that can be deployed across health and research institutions.
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http://dx.doi.org/10.1016/j.xhgg.2021.100035DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8432593PMC
July 2021

A homozygous stop-gain variant in ARHGAP42 is associated with childhood interstitial lung disease, systemic hypertension, and immunological findings.

PLoS Genet 2021 07 7;17(7):e1009639. Epub 2021 Jul 7.

Division of Newborn Medicine, Boston Children's Hospital and Harvard Medical School, Boston, Massachusetts, United States of America.

ARHGAP42 encodes Rho GTPase activating protein 42 that belongs to a member of the GTPase Regulator Associated with Focal Adhesion Kinase (GRAF) family. ARHGAP42 is involved in blood pressure control by regulating vascular tone. Despite these findings, disorders of human variants in the coding part of ARHGAP42 have not been reported. Here, we describe an 8-year-old girl with childhood interstitial lung disease (chILD), systemic hypertension, and immunological findings who carries a homozygous stop-gain variant (c.469G>T, p.(Glu157Ter)) in the ARHGAP42 gene. The family history is notable for both parents with hypertension. Histopathological examination of the proband lung biopsy showed increased mural smooth muscle in small airways and alveolar septa, and concentric medial hypertrophy in pulmonary arteries. ARHGAP42 stop-gain variant in the proband leads to exon 5 skipping, and reduced ARHGAP42 levels, which was associated with enhanced RhoA and Cdc42 expression. This is the first report linking a homozygous stop-gain variant in ARHGAP42 with a chILD disorder, systemic hypertension, and immunological findings in human patient. Evidence of smooth muscle hypertrophy on lung biopsy and an increase in RhoA/ROCK signaling in patient cells suggests the potential mechanistic link between ARHGAP42 deficiency and the development of chILD disorder.
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http://dx.doi.org/10.1371/journal.pgen.1009639DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8289122PMC
July 2021

Unique variants in CLCN3, encoding an endosomal anion/proton exchanger, underlie a spectrum of neurodevelopmental disorders.

Am J Hum Genet 2021 08 28;108(8):1450-1465. Epub 2021 Jun 28.

Erasmus University Medical Center, Department of Clinical Genetics, 3000 CA Rotterdam, the Netherlands.

The genetic causes of global developmental delay (GDD) and intellectual disability (ID) are diverse and include variants in numerous ion channels and transporters. Loss-of-function variants in all five endosomal/lysosomal members of the CLC family of Cl channels and Cl/H exchangers lead to pathology in mice, humans, or both. We have identified nine variants in CLCN3, the gene encoding CIC-3, in 11 individuals with GDD/ID and neurodevelopmental disorders of varying severity. In addition to a homozygous frameshift variant in two siblings, we identified eight different heterozygous de novo missense variants. All have GDD/ID, mood or behavioral disorders, and dysmorphic features; 9/11 have structural brain abnormalities; and 6/11 have seizures. The homozygous variants are predicted to cause loss of ClC-3 function, resulting in severe neurological disease similar to the phenotype observed in Clcn3 mice. Their MRIs show possible neurodegeneration with thin corpora callosa and decreased white matter volumes. Individuals with heterozygous variants had a range of neurodevelopmental anomalies including agenesis of the corpus callosum, pons hypoplasia, and increased gyral folding. To characterize the altered function of the exchanger, electrophysiological analyses were performed in Xenopus oocytes and mammalian cells. Two variants, p.Ile607Thr and p.Thr570Ile, had increased currents at negative cytoplasmic voltages and loss of inhibition by luminal acidic pH. In contrast, two other variants showed no significant difference in the current properties. Overall, our work establishes a role for CLCN3 in human neurodevelopment and shows that both homozygous loss of ClC-3 and heterozygous variants can lead to GDD/ID and neuroanatomical abnormalities.
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http://dx.doi.org/10.1016/j.ajhg.2021.06.003DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8387284PMC
August 2021

Haploinsufficiency of ARFGEF1 is associated with developmental delay, intellectual disability, and epilepsy with variable expressivity.

Genet Med 2021 10 10;23(10):1901-1911. Epub 2021 Jun 10.

Inserm UMR1231 team GAD, University of Burgundy and Franche-Comté, Dijon, France.

Purpose: ADP ribosylation factor guanine nucleotide exchange factors (ARFGEFs) are a family of proteins implicated in cellular trafficking between the Golgi apparatus and the plasma membrane through vesicle formation. Among them is ARFGEF1/BIG1, a protein involved in axon elongation, neurite development, and polarization processes. ARFGEF1 has been previously suggested as a candidate gene for different types of epilepsies, although its implication in human disease has not been well characterized.

Methods: International data sharing, in silico predictions, and in vitro assays with minigene study, western blot analyses, and RNA sequencing.

Results: We identified 13 individuals with heterozygous likely pathogenic variants in ARFGEF1. These individuals displayed congruent clinical features of developmental delay, behavioral problems, abnormal findings on brain magnetic resonance image (MRI), and epilepsy for almost half of them. While nearly half of the cohort carried de novo variants, at least 40% of variants were inherited from mildly affected parents who were clinically re-evaluated by reverse phenotyping. Our in silico predictions and in vitro assays support the contention that ARFGEF1-related conditions are caused by haploinsufficiency, and are transmitted in an autosomal dominant fashion with variable expressivity.

Conclusion: We provide evidence that loss-of-function variants in ARFGEF1 are implicated in sporadic and familial cases of developmental delay with or without epilepsy.
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http://dx.doi.org/10.1038/s41436-021-01218-6DOI Listing
October 2021

PIGH deficiency can be associated with severe neurodevelopmental and skeletal manifestations.

Clin Genet 2021 02 27;99(2):313-317. Epub 2020 Nov 27.

Department of Pediatrics, CHU Sainte-Justine, Montreal, QC, Canada.

Phosphatidylinositol Glycan Anchor Biosynthesis class H (PIGH) is an essential player in the glycosylphosphatidylinositol (GPI) synthesis, an anchor for numerous cell membrane-bound proteins. PIGH deficiency is a newly described and rare disorder associated with developmental delay, seizures and behavioral difficulties. Herein, we report three new unrelated families with two different bi-allelic PIGH variants, including one new variant p.(Arg163Trp) which seems associated with a more severe phenotype. The common clinical features in all affected individuals are developmental delay/intellectual disability and hypotonia. Variable clinical features include seizures, autism spectrum disorder, apraxia, severe language delay, dysarthria, feeding difficulties, facial dysmorphisms, microcephaly, strabismus, and musculoskeletal anomalies. The two siblings homozygous for the p.(Arg163Trp) variant have severe symptoms including profound psychomotor retardation, intractable seizures, multiple bone fractures, scoliosis, loss of independent ambulation, and delayed myelination on brain MRI. Serum iron levels were significantly elevated in one individual. All tested individuals with PIGH deficiency had normal alkaline phosphatase and CD16, a GPI-anchored protein (GPI-AP), was found to be decreased by 60% on granulocytes from one individual. This study expands the PIGH deficiency phenotype range toward the severe end of the spectrum with the identification of a novel pathogenic variant.
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http://dx.doi.org/10.1111/cge.13877DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7839508PMC
February 2021

The P72R Polymorphism in R248Q/W p53 Mutants Modifies the Mutant Effect on Epithelial to Mesenchymal Transition Phenotype and Cell Invasion via CXCL1 Expression.

Int J Mol Sci 2020 Oct 28;21(21). Epub 2020 Oct 28.

Department of Biochemistry and Molecular Medicine, University of California Davis Medical Center, Sacramento, CA 95817, USA.

High-grade serous carcinoma (HGSC), the most lethal subtype of epithelial ovarian cancer (EOC), is characterized by widespread mutations (>90%), most of which are missense mutations (>70%). The objective of this study was to investigate differential transcriptional targets affected by a common germline P72R SNP () in two p53 hotspot mutants, R248Q and R248W, and identify the mechanism through which the P72R SNP affects the neomorphic properties of these mutants. Using isogenic cell line models, transcriptomic analysis, xenografts, and patient data, we found that the P72R SNP modifies the effect of p53 hotspot mutants on cellular morphology and invasion properties. Most importantly, RNA sequencing studies identified CXCL1 a critical factor that is differentially affected by P72R SNP in R248Q and R248W mutants and is responsible for differences in cellular morphology and functional properties observed in these p53 mutants. We show that the mutants with the P72 SNP promote a reversion of the EMT phenotype to epithelial characteristics, whereas its R72 counterpart promotes a mesenchymal transition via the chemokine CXCL1. These studies reveal a new role of the P72R SNP in modulating the neomorphic properties of p53 mutants via CXCL1, which has significant implications for tumor invasion and metastasis.
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http://dx.doi.org/10.3390/ijms21218025DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7662892PMC
October 2020

Exome sequencing identifies novel missense and deletion variants in RTN4IP1 associated with optic atrophy, global developmental delay, epilepsy, ataxia, and choreoathetosis.

Am J Med Genet A 2021 01 9;185(1):203-207. Epub 2020 Oct 9.

Division of Genetics and Genomics, Department of Pediatrics, Boston Children's Hospital, Harvard Medical School, Boston, Massachusetts, USA.

Inherited optic neuropathies (IONs) are neurodegenerative disorders characterized by optic atrophy with or without extraocular manifestations. Optic atrophy-10 (OPA10) is an autosomal recessive ION recently reported to be caused by mutations in RTN4IP1, which encodes reticulon 4 interacting protein 1 (RTN4IP1), a mitochondrial ubiquinol oxydo-reductase. Here we report novel compound heterozygous mutations in RTN4IP1 in a male proband with developmental delay, epilepsy, optic atrophy, ataxia, and choreoathetosis. Workup was notable for transiently elevated lactate and lactate-to-pyruvate ratio, brain magnetic resonance imaging with optic atrophy and T2 signal abnormalities, and a nondiagnostic initial genetic workup, including chromosomal microarray and mitochondrial panel testing. Exome sequencing identified a paternally inherited missense variant (c.263T>G, p.Val88Gly) predicted to be deleterious and a maternally inherited deletion encompassing RTN4IP1. To our knowledge, this is the first report of a non-single nucleotide pathogenic variant associated with OPA10. This case highlights the expanding phenotypic spectrum of OPA10, the association between "syndromic" cases and severe RTN4IP1 mutations, and the importance of nonbiased genetic testing, such as ES, to analyze multiple genes and variants types, in patients suspected of having genetic disease.
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http://dx.doi.org/10.1002/ajmg.a.61910DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8388561PMC
January 2021

Prospective, phenotype-driven selection of critically ill neonates for rapid exome sequencing is associated with high diagnostic yield.

Genet Med 2020 04 29;22(4):736-744. Epub 2019 Nov 29.

Division of Genetics and Genomics, Boston Children's Hospital, Boston, MA, USA.

Purpose: To investigate the impact of rapid-turnaround exome sequencing in critically ill neonates using phenotype-based subject selection criteria.

Methods: Intensive care unit babies aged <6 months with hypotonia, seizures, a complex metabolic phenotype, and/or multiple congenital malformations were prospectively enrolled for rapid (<7 day) trio-based exome sequencing. Genomic variants relevant to the presenting phenotype were returned to the medical team.

Results: A genetic diagnosis was attained in 29 of 50 (58%) sequenced cases. Twenty-seven (54%) patients received a molecular diagnosis involving known disease genes; two additional cases (4%) were solved with pathogenic variants found in novel disease genes. In 24 of the solved cases, diagnosis had impact on patient management and/or family members. Management changes included shift to palliative care, medication changes, involvement of additional specialties, and the consideration of new experimental therapies.

Conclusion: Phenotype-based patient selection is effective at identifying critically ill neonates with a high likelihood of receiving a molecular diagnosis via rapid-turnaround exome sequencing, leading to faster and more accurate diagnoses, reducing unnecessary testing and procedures, and informing medical care.
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http://dx.doi.org/10.1038/s41436-019-0708-6DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7127968PMC
April 2020

Infant mortality: the contribution of genetic disorders.

J Perinatol 2019 12 8;39(12):1611-1619. Epub 2019 Aug 8.

Division of Newborn Medicine, Department of Medicine, Boston Children's Hospital, Harvard Medical School, Boston, MA, 02115, USA.

Objective: To determine the proportion of infant deaths occurring in the setting of a confirmed genetic disorder.

Study Design: A retrospective analysis of the electronic medical records of infants born from 1 January, 2011 to 1 June, 2017, who died prior to 1 year of age.

Results: Five hundred and seventy three deceased infants were identified. One hundred and seventeen were confirmed to have a molecular or cytogenetic diagnosis in a clinical diagnostic laboratory and an additional seven were diagnosed by research testing for a total of 124/573 (22%) diagnosed infants. A total of 67/124 (54%) had chromosomal disorders and 58/124 (47%) had single gene disorders (one infant had both). The proportion of diagnoses made by sequencing technologies, such as exome sequencing, increased over the years.

Conclusions: The prevalence of confirmed genetic disorders within our cohort of infant deaths is higher than that previously reported. Increased efforts are needed to further understand the mortality burden of genetic disorders in infancy.
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http://dx.doi.org/10.1038/s41372-019-0451-5DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6879816PMC
December 2019

Unique bioinformatic approach and comprehensive reanalysis improve diagnostic yield of clinical exomes.

Eur J Hum Genet 2019 09 12;27(9):1398-1405. Epub 2019 Apr 12.

Division of Newborn Medicine and Neonatal Genomics Program, Boston Children's Hospital, Harvard Medical School, Boston, MA, 02115, USA.

Clinical exome sequencing (CES) is increasingly being utilized; however, a large proportion of patients remain undiagnosed, creating a need for a systematic approach to increase the diagnostic yield. We have reanalyzed CES data for a clinically heterogeneous cohort of 102 probands with likely Mendelian conditions, including 74 negative cases and 28 cases with candidate variants, but reanalysis requested by clinicians. Reanalysis was performed by an interdisciplinary team using a validated custom-built pipeline, "Variant Explorer Pipeline" (VExP). This reanalysis approach and results were compared with existing literature. Reanalysis of candidate variants from CES in 28 cases revealed 1 interpretation that needed to be reclassified. A confirmed or potential genetic diagnosis was identified in 24 of 75 CES-negative/reclassified cases (32.0%), including variants in known disease-causing genes (n = 6) or candidate genes (n = 18). This yield was higher compared with similar studies demonstrating the utility of this approach. In summary, reanalysis of negative CES in a research setting enhances diagnostic yield by about a third. This study suggests the need for comprehensive, continued reanalysis of exome data when molecular diagnosis is elusive.
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http://dx.doi.org/10.1038/s41431-019-0401-xDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6777619PMC
September 2019

Olaparib-induced Adaptive Response Is Disrupted by FOXM1 Targeting that Enhances Sensitivity to PARP Inhibition.

Mol Cancer Res 2018 06 15;16(6):961-973. Epub 2018 Mar 15.

Department of Internal Medicine, University of New Mexico Comprehensive Cancer Center, University of New Mexico School of Medicine, Albuquerque, New Mexico.

FOXM1 transcription factor network is activated in over 84% of cases in high-grade serous ovarian cancer (HGSOC), and FOXM1 upregulates the expression of genes involved in the homologous recombination (HR) DNA damage and repair (DDR) pathway. However, the role of FOXM1 in PARP inhibitor response has not yet been studied. This study demonstrates that PARP inhibitor (PARPi), olaparib, induces the expression and nuclear localization of FOXM1. On the basis of ChIP-qPCR, olaparib enhances the binding of FOXM1 to genes involved in HR repair. FOXM1 knockdown by RNAi or inhibition by thiostrepton decreases FOXM1 expression, decreases the expression of HR repair genes, such as and , and enhances sensitivity to olaparib. Comet and PARP trapping assays revealed increases in DNA damage and PARP trapping in FOXM1-inhibited cells treated with olaparib. Finally, thiostrepton decreases the expression of BRCA1 in rucaparib-resistant cells and enhances sensitivity to rucaparib. Collectively, these results identify that FOXM1 plays an important role in the adaptive response induced by olaparib and FOXM1 inhibition by thiostrepton induces "BRCAness" and enhances sensitivity to PARP inhibitors. FOXM1 inhibition represents an effective strategy to overcome resistance to PARPi, and targeting FOXM1-mediated adaptive pathways may produce better therapeutic effects for PARP inhibitors. .
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http://dx.doi.org/10.1158/1541-7786.MCR-17-0607DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6705145PMC
June 2018

Progressive obesity alters ovarian insulin, phosphatidylinositol-3 kinase, and chemical metabolism signaling pathways and potentiates ovotoxicity induced by phosphoramide mustard in mice.

Biol Reprod 2017 02;96(2):478-490

Department of Animal Science, 2356 Kildee Hall, Iowa State University, Ames, IA, USA.

Mechanisms underlying obesity-associated reproductive impairment are ill defined. Hyperinsulinemia is a metabolic perturbation often observed in obese subjects. Insulin activates phosphatidylinositol 3-kinase (PI3K) signaling, which regulates ovarian folliculogenesis, steroidogenesis, and xenobiotic metabolism. The impact of progressive obesity on ovarian genes encoding mRNA involved in insulin-mediated PI3K signaling and xenobiotic biotransformation [insulin receptor (Insr), insulin receptor substrate 1 (Irs1), 2 (Irs2), and 3 (Irs3); kit ligand (Kitlg), stem cell growth factor receptor (Kit), protein kinase B (AKT) alpha (Akt1), beta (Akt2), forkhead transcription factor (FOXO) subfamily 1 (Foxo1), and subfamily 3 (Foxo3a), microsomal epoxide hydrolase (Ephx1), cytochrome P450 family 2, subfamily E, polypeptide 1 (Cyp2e1), glutathione S-transferase (GST) class Pi (Gstp1) and class mu 1 (Gstm1)] was determined in normal wild-type nonagouti (a/a; lean) and lethal yellow mice (KK.CG-Ay/J; obese) at 6, 12, 18, or 24 weeks of age. At 6 weeks, ovaries from obese mice had increased (P < 0.05) Insr and Irs3 but decreased (P < 0.05) Kitlg, Foxo1, and Cyp2e1 mRNA levels. Interestingly, at 12 weeks, an increase (P < 0.05) in Kitlg and Kit mRNA, pIRS1Ser302, pAKTThr308, EPHX1, and GSTP1 protein level was observed due to obesity, while Cyp2e1 mRNA and protein were reduced. A phosphoramide mustard (PM) challenge increased (P < 0.05) ovarian EPHX1 protein abundance in lean but not obese females. In addition, lung tissue from PM-exposed animals had increased (P < 0.05) EPHX1 protein with no impact of obesity thereon. Taken together, progressive obesity affected ovarian signaling pathways potentially involved in obesity-associated reproductive disorders.
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http://dx.doi.org/10.1095/biolreprod.116.143818DOI Listing
February 2017

Obesity alters phosphoramide mustard-induced ovarian DNA repair in mice.

Biol Reprod 2017 02;96(2):491-501

Department of Animal Science, Iowa State University, Ames, IA, USA.

Phosphoramide mustard (PM) destroys rapidly dividing cells and activates the DNA double strand break marker, γH2AX, and DNA repair in rat granulosa cells and neonatal ovaries. The effects of PM exposure on DNA damage and activation of DNA damage repair in lean and obese female mice were investigated. Wild type (lean) non agouti (a/a) and KK.Cg-Ay/J heterozygote (obese) mice received sesame oil or PM (95%; 25 mg/kg; intraperitoneal injection). Obesity increased (P < 0.05) hepatic and spleen but decreased (P < 0.05) uterine weight. PM exposure reduced (P < 0.05) spleen weight regardless of body composition, however, decreased (P < 0.05) ovarian and hepatic weight were observed in the obese PM-exposed females. PM decreased (P < 0.05) primordial and primary follicle number in lean females. Obesity and PM increased (P < 0.05) γH2AX protein. DNA damage repair genes Prkdc, Parp1, and Rad51 mRNA were unaltered by obesity, however, Atm and Xrcc6 mRNA were increased (P < 0.05) while Brca1 was reduced (P < 0.05). Obesity reduced (P < 0.05) PRKDC, XRCC6 and but increased (P < 0.05) ATM protein. ATM, BRCA1 and RAD51 protein levels were increased (P < 0.05) by PM exposure in both lean and obese mice, while PM-induced increased (P < 0.05) XRCC6 and PARP1 were observed only in lean mice. Thus, PM induces ovarian DNA damage in vivo; obesity alters DNA repair response gene mRNA and protein level; the ovary activates DNA repair proteins in response to PM; but obesity compromises the ovarian PM response.
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http://dx.doi.org/10.1095/biolreprod.116.143800DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6366544PMC
February 2017

Phosphoramide mustard induces autophagy markers and mTOR inhibition prevents follicle loss due to phosphoramide mustard exposure.

Reprod Toxicol 2017 01 22;67:65-78. Epub 2016 Nov 22.

Department of Animal Science, Iowa State University, Ames, IA, 50011, United States. Electronic address:

Phosphoramide mustard (PM) is an ovotoxic metabolite of cyclophosphamide. Postnatal day 4 Fisher 344 rat ovaries were exposed to vehicle control (1% DMSO) or PM (60μM)±LY294002 or rapamycin for 2 or 4 d. Transmission election microscopy revealed abnormally large golgi apparatus and electron dense mitochondria in PM-exposed ovaries prior to and at the time of follicle depletion. PM exposure increased (P<0.05) mRNA abundance of Bbc3, Cdkn1a, Ctfr, Edn1, Gstp1, Nqo1, Tlr4, Tnfrsfla, Txnrd1 and decreased (P<0.05) Casp1 and Il1b after 4d. PM exposure increased (P<0.1) BECN1 and LAMP, decreased (P<0.1) ABCB1 and did not alter ABCC1 protein. LY294002 did not impact PM-induced ovotoxicity, but decreased ABCC1 and ABCB1 protein. Rapamycin prevented PM-induced follicle loss. These data suggest that the mammalian target of rapamycin, mTOR, may be a gatekeeper of PM-induced follicle loss.
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http://dx.doi.org/10.1016/j.reprotox.2016.11.014DOI Listing
January 2017

Ovarian xenobiotic biotransformation enzymes are altered during phosphoramide mustard-induced ovotoxicity.

Toxicol Sci 2014 Oct 28;141(2):441-52. Epub 2014 Jul 28.

Department of Animal Science, Iowa State University, Ames, Iowa 50011

The anti-neoplastic prodrug, cyclophosphamide, requires biotransformation to phosphoramide mustard (PM), which partitions to volatile chloroethylaziridine (CEZ). PM and CEZ are ovotoxicants, however their ovarian biotransformation remains ill-defined. This study investigated PM and CEZ metabolism mechanisms through the utilization of cultured postnatal day 4 (PND4) Fisher 344 (F344) rat ovaries exposed to vehicle control (1% dimethyl sulfoxide (DMSO)) or PM (60μM) for 2 or 4 days. Quantification of mRNA levels via an RT(2) profiler PCR array and target-specific RT-PCR along with Western blotting found increased mRNA and protein levels of xenobiotic metabolism genes including microsomal epoxide hydrolase (Ephx1) and glutathione S-transferase isoform pi (Gstp). PND4 ovaries were treated with 1% DMSO, PM (60μM), cyclohexene oxide to inhibit EPHX1 (CHO; 2mM), or PM + CHO for 4 days. Lack of functional EPHX1 increased PM-induced ovotoxicity, suggesting a detoxification role for EPHX1. PND4 ovaries were also treated with 1% DMSO, PM (60μM), BSO (Glutathione (GSH) depletion; 100μM), GEE (GSH supplementation; 2.5mM), PM ± BSO, or PM ± GEE for 4 days. GSH supplementation prevented PM-induced follicle loss, whereas no impact of GSH depletion was observed. Lastly, the effect of ovarian GSH on CEZ liberation and ovotoxicity was evaluated. Both untreated and GEE-treated PND4 ovaries were plated adjacent to ovaries receiving PM + GEE or PM + BSO treatments. Less CEZ-induced ovotoxicity was observed with both GEE and BSO treatments indicating reduced CEZ liberation from PM. Collectively, this study supports ovarian biotransformation of PM, thereby influencing the ovotoxicity that ensues.
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http://dx.doi.org/10.1093/toxsci/kfu146DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4271048PMC
October 2014

Involvement of a volatile metabolite during phosphoramide mustard-induced ovotoxicity.

Toxicol Appl Pharmacol 2014 May 15;277(1):1-7. Epub 2014 Mar 15.

Department of Animal Science, Iowa State University, Ames, IA 50011, USA; Department of Physiology, University of Arizona, Tucson, AZ 85724, USA. Electronic address:

The finite ovarian follicle reserve can be negatively impacted by exposure to chemicals including the anti-neoplastic agent, cyclophosphamide (CPA). CPA requires bioactivation to phosphoramide mustard (PM) to elicit its therapeutic effects however; in addition to being the tumor-targeting metabolite, PM is also ovotoxic. In addition, PM can break down to a cytotoxic, volatile metabolite, chloroethylaziridine (CEZ). The aim of this study was initially to characterize PM-induced ovotoxicity in growing follicles. Using PND4 Fisher 344 rats, ovaries were cultured for 4 days before being exposed once to PM (10 or 30 μM). Following eight additional days in culture, relative to control (1% DMSO), PM had no impact on primordial, small primary or large primary follicle number, but both PM concentrations induced secondary follicle depletion (P<0.05). Interestingly, a reduction in follicle number in the control-treated ovaries was observed. Thus, the involvement of a volatile, cytotoxic PM metabolite (VC) in PM-induced ovotoxicity was explored in cultured rat ovaries, with control ovaries physically separated from PM-treated ovaries during culture. Direct PM (60 μM) exposure destroyed all stage follicles after 4 days (P<0.05). VC from nearby wells depleted primordial follicles after 4 days (P<0.05), temporarily reduced secondary follicle number after 2 days, and did not impact other stage follicles at any other time point. VC was determined to spontaneously liberate from PM, which could contribute to degradation of PM during storage. Taken together, this study demonstrates that PM and VC are ovotoxicants, with different follicular targets, and that the VC may be a major player during PM-induced ovotoxicity observed in cancer survivors.
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http://dx.doi.org/10.1016/j.taap.2014.03.006DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4077164PMC
May 2014

Acute 7,12-dimethylbenz[a]anthracene exposure causes differential concentration-dependent follicle depletion and gene expression in neonatal rat ovaries.

Toxicol Appl Pharmacol 2014 May 24;276(3):179-87. Epub 2014 Feb 24.

Department of Animal Science, Iowa State University, Ames, IA 50011, USA; Department of Physiology, University of Arizona, Tucson, AZ 85724, USA. Electronic address:

Chronic exposure to the polycyclic aromatic hydrocarbon 7,12-dimethylbenz[a]anthracene (DMBA), generated during combustion of organic matter including cigarette smoke, depletes all ovarian follicle types in the mouse and rat, and in vitro models mimic this effect. To investigate the mechanisms involved in follicular depletion during acute DMBA exposure, two concentrations of DMBA at which follicle depletion has (75 nM) and has not (12.5 nM) been observed were investigated. Postnatal day four F344 rat ovaries were maintained in culture for four days before a single exposure to vehicle control (1% DMSO; CT) or DMBA (12 nM; low-concentration or 75 nM; high-concentration). After four or eight additional days of culture, DMBA-induced follicle depletion was evaluated via follicle enumeration. Relative to control, DMBA did not affect follicle numbers after 4 days of exposure, but induced large primary follicle loss at both concentrations after 8 days; while, the low-concentration DMBA also caused secondary follicle depletion. Neither concentration affected primordial or small primary follicle number. RNA was isolated and quantitative RT-PCR performed prior to follicle loss to measure mRNA levels of genes involved in xenobiotic metabolism (Cyp2e1, Gstmu, Gstpi, Ephx1), autophagy (Atg7, Becn1), oxidative stress response (Sod1, Sod2) and the phosphatidylinositol 3-kinase (PI3K) pathway (Kitlg, cKit, Akt1) 1, 2 and 4 days after exposure. With the exception of Atg7 and cKit, DMBA increased (P < 0.05) expression of all genes investigated. Also, BECN1 and pAKT(Thr308) protein levels were increased while cKIT was decreased by DMBA exposure. Taken together, these results suggest an increase in DMBA bioactivation, add to the mechanistic understanding of DMBA-induced ovotoxicity and raise concern regarding female low concentration DMBA exposures.
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http://dx.doi.org/10.1016/j.taap.2014.02.011DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4077181PMC
May 2014

Glutathione S-transferase class μ regulation of apoptosis signal-regulating kinase 1 protein during VCD-induced ovotoxicity in neonatal rat ovaries.

Toxicol Appl Pharmacol 2013 Feb 27;267(1):49-56. Epub 2012 Dec 27.

Department of Animal Science, Iowa State University, Ames, IA 50011, USA.

4-Vinylcyclohexene diepoxide (VCD) destroys ovarian primordial and small primary follicles via apoptosis. In mice, VCD exposure induces ovarian mRNA expression of glutathione S-transferase (GST) family members, including isoform mu (Gstm). Extra-ovarian GSTM negatively regulates pro-apoptotic apoptosis signal-regulating kinase 1 (ASK1) through protein complex formation, which dissociates during stress, thereby initiating ASK1-induced apoptosis. The present study investigated the ovarian response of Gstm mRNA and protein to VCD. Induction of Ask1 mRNA at VCD-induced follicle loss onset was determined. Ovarian GSTM:ASK1 protein complex formation was investigated and VCD exposure effects thereon evaluated. Phosphatidylinositol-3 kinase (PI3K) regulation of GSTM protein was also studied. Postnatal day (PND) 4 rat ovaries were cultured in control media ± 1) VCD (30 μM) for 2-8 days; 2) VCD (30 μM) for 2 days, followed by incubation in control media for 4 days (acute VCD exposure); or 3) LY294002 (20 μM) for 6 days. VCD exposure did not alter Gstm mRNA expression, however, GSTM protein increased (P<0.05) after 6 days of both the acute and chronic treatments. Ask1 mRNA increased (0.33-fold; P<0.05) relative to control after 6 days of VCD exposure. Ovarian GSTM:ASK1 protein complex formation was confirmed and, relative to control, the amount of GSTM bound to ASK1 increased 33% (P<0.05) by chronic but with no effect of acute VCD exposure. PI3K inhibition increased (P<0.05) GSTM protein by 40% and 71% on d4 and d6, respectively. These findings support involvement of GSTM in the ovarian response to VCD exposure, through regulation of pro-apoptotic ASK1.
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http://dx.doi.org/10.1016/j.taap.2012.12.013DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3575192PMC
February 2013

Rates of molecular evolution vary in vertebrates for insulin-like growth factor-1 (IGF-1), a pleiotropic locus that regulates life history traits.

Gen Comp Endocrinol 2012 Aug 30;178(1):164-73. Epub 2012 Apr 30.

Department of Ecology, Evolution & Organismal Biology, Iowa State University, Ames, IA 50011, USA.

Insulin-like growth factor-1 (IGF-1) is a member of the vertebrate insulin/insulin-like growth factor/relaxin gene family necessary for growth, reproduction, and survival at both the cellular and organismal level. Its sequence, protein structure, and function have been characterized in mammals, birds, and fish; however, a notable gap in our current knowledge of the function of IGF-1 and its molecular evolution is information in ectothermic reptiles. To address this disparity, we sequenced the coding region of IGF-1 in 11 reptile species-one crocodilian, three turtles, three lizards, and four snakes. Complete sequencing of the full mRNA transcript of a snake revealed the Ea-isoform, the predominant isoform of IGF-1 also reported in other vertebrate groups. A gene tree of the IGF-1 protein-coding region that incorporated sequences from diverse vertebrate groups showed similarity to the species phylogeny, with the exception of the placement of Testudines as sister group to Aves, due to their high nucleotide sequence similarity. In contrast, long-branch lengths indicate more rapid divergence in IGF-1 among lizards and snakes. Additionally, lepidosaurs (i.e., lizards and snakes) had higher rates of non-synonymous:synonymous substitutions (dN/dS) relative to archosaurs (i.e., birds and crocodilians) and turtles. Tests for positive selection on specific codons within branches and evaluation of the changes in the amino acid properties, suggested positive selection in lepidosaurs on the C domain of IGF-1, which is involved in binding affinity to the IGF-1 receptor. Predicted structural changes suggest that major alterations in protein structure and function may have occurred in reptiles. These data propose new insights into the molecular co-evolution of IGF-1 and its receptors, and ultimately the evolution of IGF-1's role in regulating life-history traits across vertebrates.
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http://dx.doi.org/10.1016/j.ygcen.2012.04.022DOI Listing
August 2012
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