Publications by authors named "Patricia Donahoe"

104 Publications

Rare and de novo variants in 827 congenital diaphragmatic hernia probands implicate LONP1 as candidate risk gene.

Am J Hum Genet 2021 10 20;108(10):1964-1980. Epub 2021 Sep 20.

Department of Surgery, Boston Children's Hospital, Boston, MA 02115, USA.

Congenital diaphragmatic hernia (CDH) is a severe congenital anomaly that is often accompanied by other anomalies. Although the role of genetics in the pathogenesis of CDH has been established, only a small number of disease-associated genes have been identified. To further investigate the genetics of CDH, we analyzed de novo coding variants in 827 proband-parent trios and confirmed an overall significant enrichment of damaging de novo variants, especially in constrained genes. We identified LONP1 (lon peptidase 1, mitochondrial) and ALYREF (Aly/REF export factor) as candidate CDH-associated genes on the basis of de novo variants at a false discovery rate below 0.05. We also performed ultra-rare variant association analyses in 748 affected individuals and 11,220 ancestry-matched population control individuals and identified LONP1 as a risk gene contributing to CDH through both de novo and ultra-rare inherited largely heterozygous variants clustered in the core of the domains and segregating with CDH in affected familial individuals. Approximately 3% of our CDH cohort who are heterozygous with ultra-rare predicted damaging variants in LONP1 have a range of clinical phenotypes, including other anomalies in some individuals and higher mortality and requirement for extracorporeal membrane oxygenation. Mice with lung epithelium-specific deletion of Lonp1 die immediately after birth, most likely because of the observed severe reduction of lung growth, a known contributor to the high mortality in humans. Our findings of both de novo and inherited rare variants in the same gene may have implications in the design and analysis for other genetic studies of congenital anomalies.
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http://dx.doi.org/10.1016/j.ajhg.2021.08.011DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8546037PMC
October 2021

Urinary phthalate metabolite concentrations are negatively associated with follicular fluid anti-müllerian hormone concentrations in women undergoing fertility treatment.

Environ Int 2021 12 7;157:106809. Epub 2021 Aug 7.

Department of Environmental Health, Harvard T.H. Chan School of Public Health, Boston, MA, United States; Channing Division of Network Medicine, Harvard Medical School & Brigham and Women's Hospital, United States.

Exposure to phthalates, endocrine-disrupting chemicals commonly used as plasticizers and in consumer products, has been associated with infertility and premature ovarian failure. Our objective was to investigate whether urinary phthalate metabolite concentrations were associated with pre-ovulatory follicular fluid (FF) anti-müllerian hormone (AMH) concentrations in women undergoing fertility treatment. This cross-sectional analysis included 138 women with urinary phthalate data available in the Environment and Reproductive Health (EARTH) Study (2010-2016) in whom FF AMH concentrations were quantified using a sandwich enzyme-linked immunosorbent assay (ELISA). We also quantified 8 phthalate metabolite concentrations using tandem mass spectrometry in 1-2 urine samples per cycle (total 331 urines) and calculated the cycle-specific geometric mean for each metabolite. We applied cluster-weighted generalized estimating equation models (CWGEE) to evaluate the associations of tertiles of urinary phthalate metabolite concentrations with log-transformed FF AMH concentrations adjusting for potential confounders. Study participants had median age of 34.0 years (IQR 32.0, 37.0), 83% were white, and median BMI of 23.1 kg/m (IQR 21.2, 26.1). The following stimulation protocols were used: luteal phase agonist (70%), antagonist (14%), or flare (16%). Urinary concentrations of select phthalate metabolites were negatively associated with FF AMH. For example, women whose urinary mEOHP was in the lowest tertile (range 0.30-4.04 ng/ml) had an adjusted mean FF AMH of 0.72 ng/mL (95% CI = 0.36, 1.44), compared to women in the highest tertile (range 9.90-235), who had an adjusted mean of 0.24 ng/mL (95% CI = 0.12-0.48, p < 0.05). The negative association between urinary concentrations of certain phthalate metabolites with FF AMH concentrations may have implications for antral follicle recruitment and fertility treatment outcomes.
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http://dx.doi.org/10.1016/j.envint.2021.106809DOI Listing
December 2021

Structure of AMH bound to AMHR2 provides insight into a unique signaling pair in the TGF-β family.

Proc Natl Acad Sci U S A 2021 06;118(26)

Department of Molecular Genetics, Biochemistry, and Microbiology, University of Cincinnati, Cincinnati, OH 45267;

Anti-Müllerian hormone (AMH), or Müllerian-inhibiting substance, is a protein hormone that promotes Müllerian duct regression during male fetal sexual differentiation and regulation of folliculogenesis in women. AMH is a member of the transforming growth factor beta (TGF-β) family, which has evolved to signal through its own dedicated type II receptor, AMH receptor type II (AMHR2). Structures of other TGF-β family members have revealed how ligands infer specificity for their cognate receptors; however, it is unknown how AMH binds AMHR2 at the molecular level. Therefore, in this study, we solved the X-ray crystal structure of AMH bound to the extracellular domain of AMHR2 to a resolution of 2.6Å. The structure reveals that while AMH binds AMHR2 in a similar location to Activin and BMP ligand binding to their type II receptors, differences in both AMH and AMHR2 account for a highly specific interaction. Furthermore, using an AMH responsive cell-based luciferase assay, we show that a conformation in finger 1 of AMHR2 and a salt bridge formed by K534 on AMH and D81/E84 of AMHR2 are key to the AMH/AMHR2 interaction. Overall, our study highlights how AMH engages AMHR2 using a modified paradigm of receptor binding facilitated by modifications to the three-finger toxin fold of AMHR2. Furthermore, understanding these elements contributing to the specificity of binding will help in the design of agonists or antagonists or the selection of antibody therapies.
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http://dx.doi.org/10.1073/pnas.2104809118DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8256043PMC
June 2021

Single-cell sequencing reveals suppressive transcriptional programs regulated by MIS/AMH in neonatal ovaries.

Proc Natl Acad Sci U S A 2021 05;118(20)

Pediatric Surgical Research Laboratories, Massachusetts General Hospital, Boston, MA 02114;

Müllerian inhibiting substance (MIS/AMH), produced by granulosa cells of growing follicles, is an important regulator of folliculogenesis and follicle development. Treatment with exogenous MIS in mice suppresses follicle development and prevents ovulation. To investigate the mechanisms by which MIS inhibits follicle development, we performed single-cell RNA sequencing of whole neonatal ovaries treated with MIS at birth and analyzed at postnatal day 6, coinciding with the first wave of follicle growth. We identified distinct transcriptional signatures associated with MIS responses in the ovarian cell types. MIS treatment inhibited proliferation in granulosa, surface epithelial, and stromal cell types of the ovary and elicited a unique signature of quiescence in granulosa cells. In addition to decreasing the number of growing preantral follicles, we found that MIS treatment uncoupled the maturation of germ cells and granulosa cells. In conclusion, MIS suppressed neonatal follicle development by inhibiting proliferation, imposing a quiescent cell state, and preventing granulosa cell differentiation.
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http://dx.doi.org/10.1073/pnas.2100920118DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8157966PMC
May 2021

Follicular fluid anti-Müllerian hormone (AMH) concentrations and outcomes of in vitro fertilization cycles with fresh embryo transfer among women at a fertility center.

J Assist Reprod Genet 2020 Nov 6;37(11):2757-2766. Epub 2020 Oct 6.

Harvard T.H. Chan School of Public Health, Boston, MA, USA.

Purpose: To enhance the understanding of the clinical significance of anti-Müllerian hormone (AMH) in follicular fluid, we aimed to determine the variability of AMH concentrations in follicular fluid within and across IVF cycles and whether high follicular fluid AMH concentrations are associated with improved clinical IVF outcomes.

Methods: This was a retrospective cohort study of companion follicular fluid and serum samples from 162 women enrolled in the Environment and Reproductive Health (EARTH) Study between 2010 and 2016. AMH concentrations were quantified using a sandwich enzyme-linked immunosorbent assay. Spearman correlation and intra-class correlation (ICC) were calculated to assess variability of follicular fluid AMH, and generalized linear mixed models were used to evaluate the associations of FF AMH with IVF outcomes.

Results: The median (interquartile range, IQR) age of the 162 women was 34.0 years (32.0, 37.0). Follicular fluid AMH concentrations were highly correlated between follicles within each IVF cycle (Spearman r = 0.78 to 0.86) and across cycles for each woman (ICC 0.87 (95% CI 0.81 to 0.92)). Compared with women in the highest tertile of FF AMH (mean AMH = 2.3 ng/ml), women in the lowest tertile (mean AMH = 0.2 ng/ml) had lower serum AMH (T1 = 0.1 ng/ml vs. T3 = 0.6 ng/ml, p < 0.0001). In adjusted models, higher tertiles of follicular fluid AMH concentrations were associated with lower mean endometrial thickness and higher probability of clinical pregnancy.

Conclusions: Follicular fluid AMH concentrations show little variability between pre-ovulatory follicles, and higher pre-ovulatory follicular fluid AMH may predict a higher probability of clinical pregnancy.
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http://dx.doi.org/10.1007/s10815-020-01956-7DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7642031PMC
November 2020

Mutational Analysis of the Putative Anti-Müllerian Hormone (AMH) Binding Interface on its Type II Receptor, AMHR2.

Endocrinology 2020 07;161(7)

Department of Molecular Genetics, Biochemistry, and Microbiology, University of Cincinnati, Cincinnati, OH, USA.

Anti-Müllerian hormone (AMH) or Müllerian inhibiting substance is a unique member of the TGF-β family responsible for development and differentiation of the reproductive system. AMH signals through its own dedicated type II receptor, anti-Müllerian hormone receptor type II (AMHR2), providing an exclusive ligand-receptor pair within the broader TGF-β family. In this study, we used previous structural information to derive a model of AMH bound to AMHR2 to guide mutagenesis studies to identify receptor residues important for AMH signaling. Nonconserved mutations were introduced in AMHR2 and characterized in an AMH-responsive cell-based luciferase assay and native PAGE. Collectively, our results identified several residues important for AMH signaling within the putative ligand binding interface of AMHR2. Our results show that AMH engages AMHR2 at a similar interface to how activin and BMP class ligands bind the type II receptor, ACVR2B; however, there are significant molecular differences at the ligand interface of these 2 receptors, where ACVR2B is mostly hydrophobic and AMHR2 is predominately charged. Overall, this study shows that although the location of ligand binding on the receptor is similar to ACVR2A, ACVR2B, and BMPR2; AMHR2 uses unique ligand-receptor interactions to impart specificity for AMH.
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http://dx.doi.org/10.1210/endocr/bqaa066DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7286617PMC
July 2020

Antimullerian Hormone and Impending Menopause in Late Reproductive Age: The Study of Women's Health Across the Nation.

J Clin Endocrinol Metab 2020 04;105(4)

Department of Obstetrics and Gynecology, University of Colorado Medical School, Aurora, Colorado.

Background: A test that helps predict the time to the final menstrual period (FMP) has been sought for many years.

Objective: To assess the ability of antimullerian hormone (AMH) measurements to predictions the time to FMP.

Design: Prospective longitudinal cohort study.

Setting: The Study of Women's Health Across the Nation.

Participants And Measurements: AMH and FSH were measured in 1537 pre- or early perimenopausal women, mean age 47.5 ± 2.6 years at baseline, then serially until 12 months of amenorrhea occurred. AMH was measured using a 2-site ELISA with a detection limit of 1.85 pg/mL.

Main Outcome Measure: Areas under the receiver operating curves (AUC) for AMH-based and FSH-based predictions of time to FMP, stratified by age. Probabilities that women would undergo their FMP in the next 12, 24, or 36 months across a range of AMH values were assessed.

Results: AUCs for predicting that the FMP will occur within the next 24 months were significantly greater for AMH-based than FSH-based models. The probability that a woman with an AMH <10 pg/mL would undergo her FMP within the next 12 months ranged from 51% at h<48 years of age to 79% at ≥51 years. The probability that a woman with an AMH >100 pg/mL would not undergo her FMP within the next 12 months ranged from 97% in women <48 years old to 90% in women ≥51 years old.

Conclusions: AMH measurement helps estimate when a woman will undergo her FMP, and, in general, does so better than FSH.
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http://dx.doi.org/10.1210/clinem/dgz283DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7067546PMC
April 2020

Neoadjuvant Treatment With Müllerian-Inhibiting Substance Synchronizes Follicles and Enhances Superovulation Yield.

J Endocr Soc 2019 Nov 22;3(11):2123-2134. Epub 2019 Jul 22.

Pediatric Surgical Research Laboratories, Massachusetts General Hospital, Boston, Massachusetts.

Müllerian-inhibiting substance (MIS), also known as anti-Müllerian hormone, is thought to be a negative regulator of primordial follicle activation. We have previously reported that treatment with exogenous MIS can induce complete ovarian suppression within 5 weeks of treatment in mice. To investigate the kinetics of the return of folliculogenesis following the reversal of suppression, we treated animals with recombinant human MIS (rhMIS) protein for 40 days in adult female Nu/Nu mice and monitored the recovery of each follicle type over time. Following cessation of MIS therapy, secondary, and antral follicles returned within 30 days, along with the normalization of reproductive hormones, including LH, FSH, MIS, and Inhibin B. Furthermore, 30 days following MIS pretreatment, the number of antral follicles were significantly higher than controls, and superovulation with timed pregnant mare serum gonadotropin and human chorionic gonadotropin stimulation at this time point resulted in an approximately threefold increased yield of eggs. Use of the combined rhMIS-gonadotropin superovulation regimen in a diminished ovarian reserve (DOR) mouse model, created by 4-vinylcyclohexene dioxide treatment, also resulted in a twofold improvement in the yield of eggs. In conclusion, treatment with rhMIS can induce a reversible ovarian suppression, following which a rapid and synchronized large initial wave of growing follicles can be harnessed to enhance the response to superovulation. Therapies modulating MIS signaling may therefore augment the response to current ovarian stimulation protocols and could be particularly useful to women with DOR or poor responders to controlled ovarian hyperstimulation during fertilization.
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http://dx.doi.org/10.1210/js.2019-00190DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6821214PMC
November 2019

Single-cell sequencing of neonatal uterus reveals an Misr2+ endometrial progenitor indispensable for fertility.

Elife 2019 06 24;8. Epub 2019 Jun 24.

Pediatric Surgical Research Laboratories, Massachusetts General Hospital, Boston, United States.

The Mullerian ducts are the anlagen of the female reproductive tract, which regress in the male fetus in response to MIS. This process is driven by subluminal mesenchymal cells expressing Misr2, which trigger the regression of the adjacent Mullerian ductal epithelium. In females, these Misr2+ cells are retained, yet their contribution to the development of the uterus remains unknown. Here, we report that subluminal Misr2+ cells persist postnatally in the uterus of rodents, but recede by week 37 of gestation in humans. Using single-cell RNA sequencing, we demonstrate that ectopic postnatal MIS administration inhibits these cells and prevents the formation of endometrial stroma in rodents, suggesting a progenitor function. Exposure to MIS during the first six days of life, by inhibiting specification of the stroma, dysregulates paracrine signals necessary for uterine development, eventually resulting in apoptosis of the Misr2+ cells, uterine hypoplasia, and complete infertility in the adult female.
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http://dx.doi.org/10.7554/eLife.46349DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6650247PMC
June 2019

De novo variants in congenital diaphragmatic hernia identify MYRF as a new syndrome and reveal genetic overlaps with other developmental disorders.

PLoS Genet 2018 12 10;14(12):e1007822. Epub 2018 Dec 10.

Department of Systems Biology, Columbia University Medical Center, New York, New York, United States of America.

Congenital diaphragmatic hernia (CDH) is a severe birth defect that is often accompanied by other congenital anomalies. Previous exome sequencing studies for CDH have supported a role of de novo damaging variants but did not identify any recurrently mutated genes. To investigate further the genetics of CDH, we analyzed de novo coding variants in 362 proband-parent trios including 271 new trios reported in this study. We identified four unrelated individuals with damaging de novo variants in MYRF (P = 5.3x10(-8)), including one likely gene-disrupting (LGD) and three deleterious missense (D-mis) variants. Eight additional individuals with de novo LGD or missense variants were identified from our other genetic studies or from the literature. Common phenotypes of MYRF de novo variant carriers include CDH, congenital heart disease and genitourinary abnormalities, suggesting that it represents a novel syndrome. MYRF is a membrane associated transcriptional factor highly expressed in developing diaphragm and is depleted of LGD variants in the general population. All de novo missense variants aggregated in two functional protein domains. Analyzing the transcriptome of patient-derived diaphragm fibroblast cells suggest that disease associated variants abolish the transcription factor activity. Furthermore, we showed that the remaining genes with damaging variants in CDH significantly overlap with genes implicated in other developmental disorders. Gene expression patterns and patient phenotypes support pleiotropic effects of damaging variants in these genes on CDH and other developmental disorders. Finally, functional enrichment analysis implicates the disruption of regulation of gene expression, kinase activities, intra-cellular signaling, and cytoskeleton organization as pathogenic mechanisms in CDH.
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http://dx.doi.org/10.1371/journal.pgen.1007822DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6301721PMC
December 2018

Müllerian inhibiting substance/anti-Müllerian hormone as a fertility preservation agent.

Curr Opin Endocrinol Diabetes Obes 2018 12;25(6):399-405

Pediatric Surgical Research Laboratories, Massachusetts General Hospital, Boston.

Purpose Of Review: The nascent field of oncofertility is quickly gaining traction as novel experimental treatments are being developed, driving a renewed interest in Müllerian inhibiting substance (MIS) as an ovarian fertoprotectant.

Recent Findings: MIS is unique in its mechanisms of ovarian protection by virtue of acting directly on granulosa cells of primordial follicles and for being a benign reproductive hormone, with few side effects. We will explore in this review how it may be utilized to protect the ovary from chemotherapy, or to enhance ovarian tissue cryopreservation therapy. We will also examine potential mechanisms of action of MIS across multiple cell types, as well as current limitations in our understanding of the pharmacology of recombinant MIS.

Summary: The usefulness of MIS as a fertoprotectant may be dependent on the mechanisms of gonadotoxicity of each chemotherapeutic. Further investigation is needed to determine how to best deliver and combine MIS treatment to existing fertility management strategies.
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http://dx.doi.org/10.1097/MED.0000000000000442DOI Listing
December 2018

Towards international standardization of immunoassays for Müllerian inhibiting substance/anti-Müllerian hormone.

Reprod Biomed Online 2018 Nov 5;37(5):631-640. Epub 2018 Sep 5.

Biotherapeutics Division, NIBSC, South Mimms, Potters Bar Hertfordshire, UK.

Research Question: Is formulated and lyophilized, recombinant human Müllerian inhibiting substance, also known as anti-Müllerian hormone (AMH), suitable for the preparation of a WHO international standard to calibrate AMH immunoassays?

Design: The AMH content of a trial preparation, coded SS-581, was determined by five laboratories using seven immunoassay methods. Participants were requested to report the content of the preparation in terms of their method calibrators through the measurement of a minimum of five concentrations in the linear part of the dose-response curve. Participants were also asked to measure, concomitantly, a panel of six serum samples containing AMH at concentrations of 0.1-13.0 ng/ml.

Results: Across all assays, including two automated assays in development, the geometric mean content was 361.76 ng/ampoule with a geometric coefficient of variation (GCV%) of 39.95%. When measured by immunoassays that were commercially available at the time of the study, the mean content was 423.08 ng/ampoule, with a GCV% of 26.67%. The inter-method geometric means of five serum samples with an AMH concentration >0.3 ng/ml and measured concomitantly with dilutions of SS-581 varied with a range of GCV% of 14.90-22.35%, which may reflect the use of serum sample value transfer to calibrate current immunoassays, some of which use non-human AMH calibrators. The AMH in trial preparation SS-581 was shown to be biologically active in the Müllerian duct regression assay.

Conclusions: A reference material prepared using human recombinant AMH is a promising candidate for the preparation of an international standard for AMH for immunoassays calibrated to recombinant human AMH.
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http://dx.doi.org/10.1016/j.rbmo.2018.08.012DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6302068PMC
November 2018

Müllerian-Inhibiting Substance/Anti-Müllerian Hormone as a Predictor of Preterm Birth in Polycystic Ovary Syndrome.

J Clin Endocrinol Metab 2018 11;103(11):4187-4196

Division of Reproductive Endocrinology and Infertility, Department of Obstetrics and Gynecology, Massachusetts General Hospital, Boston, Massachusetts.

Context: There is increasing evidence for Müllerian-inhibiting substance (MIS)/anti-Müllerian hormone (AMH) physiologic activity in the human uterus, so it is relevant to study how MIS/AMH levels impact pregnancy.

Objective: To investigate the association of MIS/AMH levels with the risk of adverse obstetric outcomes.

Design: Retrospective cohort study.

Setting: Academic fertility center.

Patients: Women who became pregnant through in vitro fertilization between January 2012 and October 2016. Exclusion criteria were: oocyte donation, gestational carrier, multiple gestations, miscarriage before 20 weeks, or medically indicated preterm deliveries.

Interventions: None.

Main Outcome Measures: There were two primary outcomes, preterm birth and cesarean delivery for arrest of labor. Because MIS/AMH level is highly skewed by certain infertility diagnoses, the preterm birth analysis was stratified by polycystic ovary syndrome (PCOS) diagnosis, and the cesarean delivery for arrest of labor analysis was stratified by diminished ovarian reserve diagnosis. χ2, Mann-Whitney, and t tests were used as appropriate. A P value of <0.05 was considered statistically significant.

Results: Among women with PCOS, those who delivered prematurely had substantially higher MIS/AMH levels (18 vs 6.4 ng/mL, P = 0.003) than did those who delivered at term. At the highest MIS/AMH values, preterm deliveries predominated; above the 90th percentile in women with PCOS, all deliveries were premature. No effect of MIS/AMH level was observed in women without PCOS. We found no association between MIS/AMH values and cesarean delivery for labor arrest.

Conclusion: In women with PCOS, substantially elevated MIS/AMH levels are significantly associated with preterm birth, suggesting closer follow-up and further studies to elucidate the underlying mechanisms.
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http://dx.doi.org/10.1210/jc.2018-01320DOI Listing
November 2018

Quantification of Müllerian Inhibiting Substance/Anti-Müllerian Hormone polypeptide by isotope dilution mass spectrometry.

Anal Biochem 2018 11 6;560:50-55. Epub 2018 May 6.

National Institute for Biological Standards and Control, South Mimms, Potters Bar, EN6 3QG, UK.

Measurement of serum concentrations of Müllerian inhibiting substance (MIS), also known as anti-Müllerian Hormone (AMH) by immunoassay is gaining clinical acceptance and widespread use for the diagnosis of ovarian conditions and for prediction of the response to ovarian stimulation protocols as part of assisted reproductive therapies. Provision of an International Standard to harmonize immunoassay methods is required. It is desirable for the content of a future International Standard to be assigned in mass units for consistency with the units reported by current methods. Isotope dilution mass spectrometry (IDMS), a physicochemical method with traceability to the SI (Système International d'Unités) unit of mass, is a candidate approach to provide orthogonal data to support this mass assignment. Here, we report on the development of an IDMS method for quantitation of AMH using three peptides from different regions of the AMH monomer as surrogates for the measurement of AMH. We show the sensitivity and linearity of the standard peptides and demonstrate the reproducibility and consistency of the measurement amongst the three peptides for determining the AMH content in buffered preparations and in trial preparations of recombinant AMH, lyophilised in the presence of an excess of bovine casein.
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http://dx.doi.org/10.1016/j.ab.2018.05.006DOI Listing
November 2018

Systematic analysis of copy number variation associated with congenital diaphragmatic hernia.

Proc Natl Acad Sci U S A 2018 05 30;115(20):5247-5252. Epub 2018 Apr 30.

The Jackson Laboratory for Genomic Medicine, Farmington, CT 06032;

Congenital diaphragmatic hernia (CDH), characterized by malformation of the diaphragm and hypoplasia of the lungs, is one of the most common and severe birth defects, and is associated with high morbidity and mortality rates. There is growing evidence demonstrating that genetic factors contribute to CDH, although the pathogenesis remains largely elusive. Single-nucleotide polymorphisms have been studied in recent whole-exome sequencing efforts, but larger copy number variants (CNVs) have not yet been studied on a large scale in a case control study. To capture CNVs within CDH candidate regions, we developed and tested a targeted array comparative genomic hybridization platform to identify CNVs within 140 regions in 196 patients and 987 healthy controls, and identified six significant CNVs that were either unique to patients or enriched in patients compared with controls. These CDH-associated CNVs reveal high-priority candidate genes including , , and We also discuss CNVs that are present in only one patient in the cohort but have additional evidence of pathogenicity, including extremely rare large and/or de novo CNVs. The candidate genes within these predicted disease-causing CNVs form functional networks with other known CDH genes and play putative roles in DNA binding/transcription regulation and embryonic development. These data substantiate the importance of CNVs in the etiology of CDH, identify CDH candidate genes and pathways, and highlight the importance of ongoing analysis of CNVs in the study of CDH and other structural birth defects.
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http://dx.doi.org/10.1073/pnas.1714885115DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5960281PMC
May 2018

Genome-wide enrichment of damaging de novo variants in patients with isolated and complex congenital diaphragmatic hernia.

Hum Genet 2017 06 16;136(6):679-691. Epub 2017 Mar 16.

Pediatric Surgical Research Laboratories, Massachusetts General Hospital, Boston, MA, USA.

Congenital Diaphragmatic Hernia (CDH) is a common and often lethal birth defect characterized by diaphragmatic structural defects and pulmonary hypoplasia. CDH is isolated in 60% of newborns, but may also be part of a complex phenotype with additional anomalies. We performed whole exome sequencing (WES) on 87 individuals with isolated or complex CDH and on their unaffected parents, to assess the contribution of de novo mutations in the etiology of diaphragmatic and pulmonary defects and to identify new candidate genes. A combined analysis with 39 additional trios with complex CDH, previously published, revealed a significant genome-wide burden of de novo variants compared to background mutation rate and 900 control trios. We identified an increased burden of likely gene-disrupting (LGD, i.e. nonsense, frameshift, and canonical splice site) and predicted deleterious missense (D-mis) variants in complex and isolated CDH patients. Overall, an excess of predicted damaging de novo LGD and D-mis variants relative to the expected frequency contributed to 21% of complex cases and 12% of isolated CDH cases. The burden of de novo variants was higher in genes expressed in the developing mouse diaphragm and heart. Some overlap with genes responsible for congenital heart defects and neurodevelopmental disorders was observed in CDH patients within our cohorts. We propose that de novo variants contribute significantly to the development of CDH.
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http://dx.doi.org/10.1007/s00439-017-1774-yDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5453716PMC
June 2017

Müllerian inhibiting substance inhibits an ovarian cancer cell line via β-catenin interacting protein deregulation of the Wnt signal pathway.

Int J Oncol 2017 Mar 13;50(3):1022-1028. Epub 2017 Feb 13.

Department of Obstetrics and Gynecology, College of Medicine, The Catholic University of Korea, Seoul 137-140, Republic of Korea.

Müllerian inhibiting substance/anti-Müllerian hormone (MIS/AMH) has been suggested as a biotherapeutic agent in gynecological cancers that highly express the MIS/AMH type II receptors (MISRII/AMHRII) but the anticancer mechanisms by which MIS/AMH acts are not fully understood. Our experiments show that MIS/AMH inhibits ovarian cancer by deregulating the Wnt signal pathway via the β-catenin interacting protein (ICAT). MIS/AMH inhibition of ICAT by small interfering RNAs (siRNA) decreased ICAT driven ovarian cancer cell viability as measured by the methylthiazoltetrazolium assay, reversed cell cycle arrest and annexin V expression and diminished migration by scratch wound assay. Changes in expression of regulatory proteins were shown by western blotting. We determined that MIS/AMH upregulated ICAT in ovarian cancer cell line which caused decreased cell viability, cell cycle arrest and apoptosis. This effect, however, was blocked when ICAT was downregulated by siRNA. The present study demonstrates a role for ICAT in MIS/AMH mediated inhibition of the Wnt signaling pathway in ovarian cancer.
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http://dx.doi.org/10.3892/ijo.2017.3874DOI Listing
March 2017

AMH/MIS as a contraceptive that protects the ovarian reserve during chemotherapy.

Proc Natl Acad Sci U S A 2017 02 30;114(9):E1688-E1697. Epub 2017 Jan 30.

Pediatric Surgical Research Laboratories, Massachusetts General Hospital, Boston, MA 02114;

The ovarian reserve represents the stock of quiescent primordial follicles in the ovary which is gradually depleted during a woman's reproductive lifespan, resulting in menopause. Müllerian inhibiting substance (MIS) (or anti-Müllerian hormone/AMH), which is produced by granulosa cells of growing follicles, has been proposed as a negative regulator of primordial follicle activation. Here we show that long-term parenteral administration of superphysiological doses of MIS, using either an adeno-associated virus serotype 9 (AAV9) gene therapy vector or recombinant protein, resulted in a complete arrest of folliculogenesis in mice. The ovaries of MIS-treated mice were smaller than those in controls and did not contain growing follicles but retained a normal ovarian reserve. When mice treated with AAV9/MIS were paired with male breeders, they exhibited complete and permanent contraception for their entire reproductive lifespan, disrupted vaginal cycling, and hypergonadotropic hypogonadism. However, when ovaries from AAV9-MIS-treated mice were transplanted orthotopically into normal recipient mice, or when treatment with the protein was discontinued, folliculogenesis resumed, suggesting reversibility. One of the important causes of primary ovarian insufficiency is chemotherapy-induced primordial follicle depletion, which has been proposed to be mediated in part by increased activation. To test the hypothesis that MIS could prevent chemotherapy-induced overactivation, mice were given carboplatin, doxorubicin, or cyclophosphamide and were cotreated with AAV9-MIS, recombinant MIS protein, or vehicle controls. We found significantly more primordial follicles in MIS-treated animals than in controls. Thus treatment with MIS may provide a method of contraception with the unique characteristic of blocking primordial follicle activation that could be exploited to prevent the primary ovarian insufficiency often associated with chemotherapy.
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http://dx.doi.org/10.1073/pnas.1620729114DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5338508PMC
February 2017

Polygenic Causes of Congenital Diaphragmatic Hernia Produce Common Lung Pathologies.

Am J Pathol 2016 10 24;186(10):2532-43. Epub 2016 Aug 24.

Pediatric Surgical Research Laboratories, Massachusetts General Hospital, Boston, Massachusetts; Department of Surgery, Harvard Medical School, Boston, Massachusetts; Department of Pediatrics, Massachusetts General Hospital, Boston, Massachusetts; Department of Surgery, Boston Children's Hospital, Boston, Massachusetts.

Congenital diaphragmatic hernia (CDH) is one of the most common and lethal congenital anomalies, and significant evidence is available in support of a genetic contribution to its etiology, including single-gene knockout mice associated with diaphragmatic defects, rare monogenetic disorders in humans, familial aggregation, and association of CDH with chromosomal abnormalities. Structural lung defects in the form of lung hypoplasia are almost invariably seen in patients with CDH and frequently in animal models of this condition. Better understanding of the mechanisms of pulmonary defects in CDH has the potential for creating targeted therapies, particularly in postnatal stages, when therapeutics can have maximum clinical impact on the surviving cohorts. Successful treatment of CDH is dependent on the integration of human genomic and genetic data with developmental expression profiling, mouse knockouts, and gene network and pathway modeling, which have generated a large number of candidate genes and pathways for follow-up studies. In particular, defective alveolarization appears to be a common and potentially actionable phenotype in both patients and animal models.
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http://dx.doi.org/10.1016/j.ajpath.2016.07.006DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5222980PMC
October 2016

Type IV collagen drives alveolar epithelial-endothelial association and the morphogenetic movements of septation.

BMC Biol 2016 07 13;14:59. Epub 2016 Jul 13.

The Pediatric Surgical Research Laboratories, Massachusetts General Hospital, Boston, MA, 02114, USA.

Background: Type IV collagen is the main component of the basement membrane that gives strength to the blood-gas barrier (BGB). In mammals, the formation of a mature BGB occurs primarily after birth during alveologenesis and requires the formation of septa from the walls of the saccule. In contrast, in avians, the formation of the BGB occurs rapidly and prior to hatching. Mutation in basement membrane components results in an abnormal alveolar phenotype; however, the specific role of type IV collagen in regulating alveologenesis remains unknown.

Results: We have performed a microarray expression analysis in late chick lung development and found that COL4A1 and COL4A2 were among the most significantly upregulated genes during the formation of the avian BGB. Using mouse models, we discovered that mutations in murine Col4a1 and Col4a2 genes affected the balance between lung epithelial progenitors and differentiated cells. Mutations in Col4a1 derived from the vascular component were sufficient to cause defects in vascular development and the BGB. We also show that Col4a1 and Col4a2 mutants displayed disrupted myofibroblast proliferation, differentiation and migration. Lastly, we revealed that addition of type IV collagen protein induced myofibroblast proliferation and migration in monolayer culture and increased the formation of mesenchymal-epithelial septal-like structures in co-culture.

Conclusions: Our study showed that type IV collagen and, therefore the basement membrane, play fundamental roles in coordinating alveolar morphogenesis. In addition to its role in the formation of epithelium and vasculature, type IV collagen appears to be key for alveolar myofibroblast development by inducing their proliferation, differentiation and migration throughout the developing septum.
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http://dx.doi.org/10.1186/s12915-016-0281-2DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4942891PMC
July 2016

Anti-Müllerian Hormone Signaling Regulates Epithelial Plasticity and Chemoresistance in Lung Cancer.

Cell Rep 2016 07 7;16(3):657-71. Epub 2016 Jul 7.

Program in Molecular Therapeutics, Fox Chase Cancer Center, Philadelphia, PA 19111, USA; Program in Molecular and Cell Biology and Genetics, Drexel University College of Medicine, Philadelphia, PA 19129, USA. Electronic address:

Anti-Müllerian hormone (AMH) and its type II receptor AMHR2, both previously thought to primarily function in gonadal tissue, were unexpectedly identified as potent regulators of transforming growth factor (TGF-β)/bone morphogenetic protein (BMP) signaling and epithelial-mesenchymal transition (EMT) in lung cancer. AMH is a TGF-β/BMP superfamily member, and AMHR2 heterodimerizes with type I receptors (ALK2, ALK3) also used by the type II receptor for BMP (BMPR2). AMH signaling regulates expression of BMPR2, ALK2, and ALK3, supports protein kinase B-nuclear factor κB (AKT-NF-κB) and SMAD survival signaling, and influences BMP-dependent signaling in non-small cell lung cancer (NSCLC). AMH and AMHR2 are selectively expressed in epithelial versus mesenchymal cells, and loss of AMH/AMHR2 induces EMT. Independent induction of EMT reduces expression of AMH and AMHR2. Importantly, EMT associated with depletion of AMH or AMHR2 results in chemoresistance but sensitizes cells to the heat shock protein 90 (HSP90) inhibitor ganetespib. Recognition of this AMH/AMHR2 axis helps to further elucidate TGF-β/BMP resistance-associated signaling and suggests new strategies for therapeutic targeting of EMT.
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http://dx.doi.org/10.1016/j.celrep.2016.06.043DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4956518PMC
July 2016

De novo frameshift mutation in COUP-TFII (NR2F2) in human congenital diaphragmatic hernia.

Am J Med Genet A 2016 09 1;170(9):2457-61. Epub 2016 Jul 1.

Pediatric Surgical Research Laboratories, Massachusetts General Hospital, Boston, Massachusetts.

COUP-TFII (NR2F2) is mapped to the 15q26 deletion hotspot associated with the common and highly morbid congenital diaphragmatic hernia (CDH). Conditional homozygous deletions of COUP-TFII in mice result in diaphragmatic defects analogous to the human Bochdalek-type hernia phenotype. Despite evidence from animal models however, mutations in the coding sequence of COUP-TFII have not been reported in patients, prompting the speculation that additional coding or non-coding sequences in the 15q26 locus are necessary for diaphragmatic hernias to develop. In this report, we describe a case of a patient with a heterozygous de novo COUP-TFII frameshift mutation, presenting with CDH and an atrial septal defect. The p.Pro33AlafsTer77 mutation specifically disrupts protein isoform 1 which contains the DNA binding domain. In addition, we review other COUP-TFII sequence variations and deletions that have been described in cases of CDH. We conclude that COUP-TFII mutations can cause diaphragmatic hernias, and should be included in the differential diagnosis of CDH patients, particularly those with comorbid congenital heart defects. © 2016 Wiley Periodicals, Inc.
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http://dx.doi.org/10.1002/ajmg.a.37830DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5003181PMC
September 2016

CD44 Splice Variant v8-10 as a Marker of Serous Ovarian Cancer Prognosis.

PLoS One 2016 2;11(6):e0156595. Epub 2016 Jun 2.

Pediatric Surgical Research Laboratories, Massachusetts General Hospital, Department of Surgery, Boston, Massachusetts, United States of America.

CD44 is a transmembrane hyaluronic acid receptor gene that encodes over 100 different tissue-specific protein isoforms. The most ubiquitous, CD44 standard, has been used as a cancer stem cell marker in ovarian and other cancers. Expression of the epithelial CD44 variant containing exons v8-10 (CD44v8-10) has been associated with more chemoresistant and metastatic tumors in gastrointestinal and breast cancers, but its role in ovarian cancer is unknown; we therefore investigated its use as a prognostic marker in this disease. The gene expression profiles of 254 tumor samples from The Cancer Genome Atlas RNAseqV2 were analyzed for the presence of CD44 isoforms. A trend for longer survival was observed in patients with high expression of CD44 isoforms that include exons v8-10. Immunohistochemical (IHC) analysis of tumors for presence of CD44v8-10 was performed on an independent cohort of 210 patients with high-grade serous ovarian cancer using a tumor tissue microarray. Patient stratification based on software analysis of staining revealed a statistically significant increase in survival in patients with the highest levels of transmembrane protein expression (top 10 or 20%) compared to those with the lowest expression (bottom 10 and 20%) (p = 0.0181, p = 0.0262 respectively). Expression of CD44v8-10 in primary ovarian cancer cell lines was correlated with a predominantly epithelial phenotype characterized by high expression of epithelial markers and low expression of mesenchymal markers by qPCR, Western blot, and IHC. Conversely, detection of proteolytically cleaved and soluble extracellular domain of CD44v8-10 in patient ascites samples was correlated with significantly worse prognosis (p<0.05). Therefore, presence of transmembrane CD44v8-10 on the surface of primary tumor cells may be a marker of a highly epithelial tumor with better prognosis while enzymatic cleavage of CD44v8-10, as detected by presence of the soluble extracellular domain in ascites fluid, may be indicative of a more metastatic disease and worse prognosis.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0156595PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4890777PMC
July 2017

MiR-449a Affects Epithelial Proliferation during the Pseudoglandular and Canalicular Phases of Avian and Mammal Lung Development.

PLoS One 2016 18;11(2):e0149425. Epub 2016 Feb 18.

Pediatric Surgical Research Laboratories, Massachusetts General Hospital, Boston, MA, United States of America.

Congenital diaphragmatic hernia is associated with pulmonary hypoplasia and respiratory distress, which result in high mortality and morbidity. Although several transgenic mouse models of lung hypoplasia exist, the role of miRNAs in this phenotype is incompletely characterized. In this study, we assessed microRNA expression levels during the pseudoglandular to canalicular phase transition of normal human fetal lung development. At this critical time, when the distal respiratory portion of the airways begins to form, microarray analysis showed that the most significantly differentially expressed miRNA was miR-449a. Prediction algorithms determined that N-myc is a target of miR-449a and identified the likely miR-449a:N-myc binding sites, confirmed by luciferase assays and targeted mutagenesis. Functional ex vivo knock-down in organ cultures of murine embryonic lungs, as well as in ovo overexpression in avian embryonic lungs, suggested a role for miR-449a in distal epithelial proliferation. Finally, miR-449a expression was found to be abnormal in rare pulmonary specimens of human fetuses with Congenital Diaphragmatic Hernia in the pseudoglandular or canalicular phase. This study confirms the conserved role of miR-449a for proper pulmonary organogenesis, supporting the delicate balance between expansion of progenitor cells and their terminal differentiation, and proposes the potential involvement of this miRNA in human pulmonary hypoplasia.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0149425PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4758652PMC
August 2016

AAV9 delivering a modified human Mullerian inhibiting substance as a gene therapy in patient-derived xenografts of ovarian cancer.

Proc Natl Acad Sci U S A 2015 Aug 27;112(32):E4418-27. Epub 2015 Jul 27.

Pediatric Surgical Research Laboratories, Massachusetts General Hospital, Department of Surgery, Harvard Medical School, Boston, MA 02114;

To improve ovarian cancer patient survival, effective treatments addressing chemoresistant recurrences are particularly needed. Mullerian inhibiting substance (MIS) has been shown to inhibit the growth of a stem-like population of ovarian cancer cells. We have recently engineered peptide modifications to human MIS [albumin leader Q425R MIS (LRMIS)] that increase production and potency in vitro and in vivo. To test this novel therapeutic peptide, serous malignant ascites from highly resistant recurrent ovarian cancer patients were isolated and amplified to create low-passage primary cell lines. Purified recombinant LRMIS protein successfully inhibited the growth of cancer spheroids in vitro in a panel of primary cell lines in four of six patients tested. Adeno-associated virus (AAV) -delivered gene therapy has undergone a clinical resurgence with a good safety profile and sustained gene expression. Therefore, AAV9 was used as a single i.p. injection to deliver LRMIS to test its efficacy in inhibiting growth of palpable tumors in patient-derived ovarian cancer xenografts from ascites (PDXa). AAV9-LRMIS monotherapy resulted in elevated and sustained blood concentrations of MIS, which significantly inhibited the growth of three of five lethal chemoresistant serous adenocarcinoma PDXa models without signs of measurable or overt toxicity. Finally, we tested the frequency of MIS type II receptor expression in a tissue microarray of serous ovarian tumors by immunohistochemistry and found that 88% of patients bear tumors that express the receptor. Taken together, these preclinical data suggest that AAV9-LRMIS provides a potentially well-tolerated and effective treatment strategy poised for testing in patients with chemoresistant serous ovarian cancer.
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http://dx.doi.org/10.1073/pnas.1510604112DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4538667PMC
August 2015

Transcriptome analysis reveals that Müllerian inhibiting substance regulates signaling pathways that contribute to endometrial carcinogenesis.

Int J Oncol 2015 May 6;46(5):2039-46. Epub 2015 Mar 6.

Department of Obstetrics and Gynecology, College of Medicine, The Catholic University of Korea, Seoul 137-701, Republic of Korea.

Müllerian inhibiting substance (MIS) has been shown to inhibit growth of a number of tumors in vitro and/or in vivo, but the downstream pathways which it regulates are not fully understood. In the present study we show that MIS type II receptor was highly expressed in AN3CA cells, a cell line derived from human endometrial cancer cell in which MIS-treatment caused a reduction of cell viability, and induced cellular apoptosis and genes involved cell cycle arrest. To understand the genome-wide effects of MIS on gene regulation, we performed serial gene expression analyses from 0 to 96 h at 24 h intervals after treating AN3CA cells with MIS. Transcriptomic analysis of molecular changes induced by MIS identified 2,688 differentially expressed genes that were significantly up- or down-regulated during the 96 h study period. When the 2,688 differentially expressed genes were mapped to known biological processes, Wnt-, cancer-, proteolysis-, cytoskeleton-, cell cycle-, apoptosis-, and MAPK-signaling pathways emerged as the functions most significantly changed by MIS in AN3CA cells. Furthermore, western blot analysis validated that protein expression of cell cycle inhibitory genes, apoptotic protease activating factor-1 (APAF-1), β-catenin-interacting protein (ICAT), Rb related protein 130 (p130), and inhibitor of disheveled Dvl and Axin complex (IDAX), were gradually increased over the time of the study, whereas downstream cell cycle activating genes, cyclin-dependent kinase 2 (CDK2) and phospho-c-Jun were downregulated in MIS-treated AN3CA cells. These transcriptome analyses support previous observations that MIS functions as a tumor suppressor, potentially by regulating signaling pathways that could contribute to endometrial carcinogenesis, and indicating that MIS should be considered as a potential treatment for endometrial cancer.
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http://dx.doi.org/10.3892/ijo.2015.2920DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6903890PMC
May 2015

Müllerian inhibiting substance/anti-Müllerian hormone: A novel treatment for gynecologic tumors.

Obstet Gynecol Sci 2014 Sep 17;57(5):343-57. Epub 2014 Sep 17.

Pediatric Surgical Research Laboratories, Massachusetts General Hospital, Harvard Medical School, Boston, MA, USA.

Müllerian inhibiting substance (MIS), also called anti-Müllerian hormone (AMH), is a member of the transforming growth factor-β super-family of growth and differentiation response modifiers. It is produced in immature Sertoli cells in male embryos and binds to MIS/AMH receptors in primordial Müllerian ducts to cause regression of female reproductive structures that are the precursors to the fallopian tubes, the surface epithelium of the ovaries, the uterus, the cervix, and the upper third of the vagina. Because most gynecologic tumors originate from Müllerian duct-derived tissues, and since MIS/AMH causes regression of the Müllerian duct in male embryos, it is expected to inhibit the growth of gynecologic tumors. Purified recombinant human MIS/AMH causes growth inhibition of epithelial ovarian cancer cells and cell lines in vitro and in vitro via MIS receptor-mediated mechanism. Furthermore, several lines of evidence suggest that MIS/AMH inhibits proliferation in tissues and cell lines of other MIS/AMH receptor-expressing gynecologic tumors such as cervical, endometrial, breast, and in endometriosis as well. These findings indicate that bioactive MIS/AMH recombinant protein should be tested in patients against tumors expressing the MIS/AMH receptor complex, perhaps beginning with ovarian cancer because it has the worst prognosis. The molecular tools to identify MIS/AMH receptor expressing ovarian and other cancers are in place, thus, it is possible to select patients for treatment. An MIS/AMH ELISA exists to follow administered doses of MIS/AMH, as well. Clinical trials await the production of sufficient supplies of qualified recombinant human MIS/AMH for this purpose.
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http://dx.doi.org/10.5468/ogs.2014.57.5.343DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4175594PMC
September 2014

Molecular pathogenesis of congenital diaphragmatic hernia revealed by exome sequencing, developmental data, and bioinformatics.

Proc Natl Acad Sci U S A 2014 Aug 8;111(34):12450-5. Epub 2014 Aug 8.

The Pediatric Surgical Research Laboratories, Massachusetts General Hospital, Boston, MA 02114; Department of Surgery, Harvard Medical School, Boston, MA 02115; Broad Institute, Cambridge, MA 02141;

Congenital diaphragmatic hernia (CDH) is a common and severe birth defect. Despite its clinical significance, the genetic and developmental pathways underlying this disorder are incompletely understood. In this study, we report a catalog of variants detected by a whole exome sequencing study on 275 individuals with CDH. Predicted pathogenic variants in genes previously identified in either humans or mice with diaphragm defects are enriched in our CDH cohort compared with 120 size-matched random gene sets. This enrichment was absent in control populations. Variants in these critical genes can be found in up to 30.9% of individuals with CDH. In addition, we filtered variants by using genes derived from regions of recurrent copy number variations in CDH, expression profiles of the developing diaphragm, protein interaction networks expanded from the known CDH-causing genes, and prioritized genes with ultrarare and highly disruptive variants, in 11.3% of CDH patients. These strategies have identified several high priority genes and developmental pathways that likely contribute to the CDH phenotype. These data are valuable for comparison of candidate genes generated from whole exome sequencing of other CDH cohorts or multiplex kindreds and provide ideal candidates for further functional studies. Furthermore, we propose that these genes and pathways will enhance our understanding of the heterogeneous molecular etiology of CDH.
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http://dx.doi.org/10.1073/pnas.1412509111DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4151769PMC
August 2014

Induction of WNT inhibitory factor 1 expression by Müllerian inhibiting substance/antiMullerian hormone in the Müllerian duct mesenchyme is linked to Müllerian duct regression.

Dev Biol 2014 Feb 19;386(1):227-36. Epub 2013 Dec 19.

Vincent Center of Reproductive Biology, Department of Obstetrics, Gynecology, and Reproductive Biology, Massachusetts General Hospital, Boston, MA, United States; Harvard Medical School, Boston, MA, United States. Electronic address:

A key event during mammalian sexual development is regression of the Müllerian ducts (MDs) in the bipotential urogenital ridges (UGRs) of fetal males, which is caused by the expression of Müllerian inhibiting substance (MIS) in the Sertoli cells of the differentiating testes. The paracrine signaling mechanisms involved in MD regression are not completely understood, particularly since the receptor for MIS, MISR2, is expressed in the mesenchyme surrounding the MD, but regression occurs in both the epithelium and mesenchyme. Microarray analysis comparing MIS signaling competent and Misr2 knockout embryonic UGRs was performed to identify secreted factors that might be important for MIS-mediated regression of the MD. A seven-fold increase in the expression of Wif1, an inhibitor of WNT/β-catenin signaling, was observed in the Misr2-expressing UGRs. Whole mount in situ hybridization of Wif1 revealed a spatial and temporal pattern of expression consistent with Misr2 during the window of MD regression in the mesenchyme surrounding the MD epithelium that was absent in both female UGRs and UGRs knocked out for Misr2. Knockdown of Wif1 expression in male UGRs by Wif1-specific siRNAs beginning on embryonic day 13.5 resulted in MD retention in an organ culture assay, and exposure of female UGRs to added recombinant human MIS induced Wif1 expression in the MD mesenchyme. Knockdown of Wif1 led to increased expression of β-catenin and its downstream targets TCF1/LEF1 in the MD mesenchyme and to decreased apoptosis, resulting in partial to complete retention of the MD. These results strongly suggest that WIF1 secretion by the MD mesenchyme plays a role in MD regression in fetal males.
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http://dx.doi.org/10.1016/j.ydbio.2013.12.015DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4047716PMC
February 2014
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