Medical Genetics Commons^™

Cross-Contamination Of Crispr Guides And Other Unrelated Nucleotide Sequences Among Commercial Oligonucleotides, Hiroshi Arakawa, Hiromi Miura, Rolen M. Quadros, Masato Ohtsuka, Channabasavaiah B. Gurumurthy

Journal Articles: Genetics, Cell Biology & Anatomy

Custom oligonucleotides (oligos) are widely used reagents in biomedical research. Some common applications of oligos include polymerase chain reaction (PCR), sequencing, hybridization, microarray, and library construction. The reliability of oligos in such applications depends on their purity and specificity. Here, we report that commercially available oligos are frequently contaminated with nonspecific sequences (i.e. other unrelated oligonucleotides). Most of the oligos that we designed to amplify clustered regularly interspersed palindromic repeats (CRISPR) guide sequences contained nonspecific CRISPR guides. These contaminants were detected in research-grade oligos procured from eight commercial oligo-suppliers located in three different geographic regions of the world. Deep sequencing …

The Frequency Of Pathogenic Variation In The All Of Us Cohort Reveals Ancestry-Driven Disparities, Eric Venner, Karynne Patterson, Divya Kalra, Marsha M Wheeler, Yi-Ju Chen, Sara E Kalla, Bo Yuan, Jason H Karnes, Kimberly Walker, Joshua D Smith, Sean Mcgee, Aparna Radhakrishnan, Andrew Haddad, Philip E Empey, Qiaoyan Wang, Lee Lichtenstein, Diana Toledo, Gail Jarvik, Anjene Musick, Richard A Gibbs

Journal Articles

Disparities in data underlying clinical genomic interpretation is an acknowledged problem, but there is a paucity of data demonstrating it. The All of Us Research Program is collecting data including whole-genome sequences, health records, and surveys for at least a million participants with diverse ancestry and access to healthcare, representing one of the largest biomedical research repositories of its kind. Here, we examine pathogenic and likely pathogenic variants that were identified in the All of Us cohort. The European ancestry subgroup showed the highest overall rate of pathogenic variation, with 2.26% of participants having a pathogenic variant. Other ancestry groups …

Assessing Adequacy: A Meta-Analysis Of Rapid Onsite Evaluation Of Thyroid Nodules, Peter P. Issa, Christina Mccarthy, Mohammad Hussein, Aaron L. Albuck, Essam Emad, Mohamed Shama, Krzysztof Moroz, Eman Toraih, Emad Kandil

School of Medicine Faculty Publications

Introduction: Fine-needle aspiration (FNA) is the standard form of preoperative evaluation of thyroid nodule cytological status. A significant number FNAs are classified as inadequate for interpretation, requiring a repeat FNA which is potentially avoidable, costly, and delays treatment. To address these concerns and maximize first-time FNA adequacy, rapid onsite evaluation (ROSE) of FNA specimens was introduced. Our study aims to determine the impact of ROSE on FNA adequacy. Methods: PubMed, Embase, and Web of Science were searched for primary articles assessing the adequacy of ROSE in thyroid nodules. Results: A total of 17 studies were included for a total of …

Investigation Into Cardiac Myhc-Α 334-352-Specific Tcr Transgenic Mice Reveals A Role For Cytotoxic Cd4 T Cells In The Development Of Cardiac Autoimmunity, Meghna Sur, Mahima T. Rasquinha, Kiruthiga Mone, Chandirasegaran Massilamany, Ninaad Lasrado, Channabasavaiah B. Gurumurthy, Raymond A Sobel, Jay Reddy

Journal Articles: Genetics, Cell Biology & Anatomy

Myocarditis is one of the major causes of heart failure in children and young adults and can lead to dilated cardiomyopathy. Lymphocytic myocarditis could result from autoreactive CD4⁺ and CD8⁺ T cells, but defining antigen specificity in disease pathogenesis is challenging. To address this issue, we generated T cell receptor (TCR) transgenic (Tg) C57BL/6J mice specific to cardiac myosin heavy chain (Myhc)-α 334-352 and found that Myhc-α-specific TCRs were expressed in both CD4⁺ and CD8⁺ T cells. To investigate if the phenotype is more pronounced in a myocarditis-susceptible genetic background, we backcrossed with A/J mice. At …

Targeted Insertion Of Conditional Expression Cassettes Into The Mouse Genome Using The Modified I-Pitt, Hiromi Miura, Ayaka Nakamura, Aki Kurosaki, Ai Kotani, Masaru Motojima, Keiko Tanaka, Shigeru Kakuta, Sanae Ogiwara, Yuhsuke Ohmi, Hirotaka Komaba, Samantha L. P. Schilit, Cynthia C. Morton, Channabasavaiah B. Gurumurthy, Masato Ohtsuka

Journal Articles: Genetics, Cell Biology & Anatomy

BACKGROUND: Transgenic (Tg) mice are widely used in biomedical research, and they are typically generated by injecting transgenic DNA cassettes into pronuclei of one-cell stage zygotes. Such animals often show unreliable expression of the transgenic DNA, one of the major reasons for which is random insertion of the transgenes. We previously developed a method called "pronuclear injection-based targeted transgenesis" (PITT), in which DNA constructs are directed to insert at pre-designated genomic loci. PITT was achieved by pre-installing so called landing pad sequences (such as heterotypic LoxP sites or attP sites) to create seed mice and then injecting Cre recombinase or …

Role Of Phosphorylated Dicer1 In Tumor Progression, Raisa Reyes-Castro

Dissertations & Theses (Open Access)

DICER1 is a multidomain enzyme discovered and widely recognized for its function in small non-coding microRNA (miRNA) synthesis. In cancer development, DICER1 functions as a haploinsufficient tumor suppressor which regulates miRNAs and Epithelial-to-Mesenchymal Transition (EMT). The Arur laboratory discovered that DICER1 is phosphorylated by active ERK and that ERK-mediated phosphorylation triggers DICER1 to translocate from the cytoplasm to the nucleus of cells in worms, mice and humans. Further, a heterozygous allele of a genetically engineered mouse model of phosphomimetic Dicer1 when combined with heterozygous Kras oncogenic background contributes to lung tumor progression in vivo. Mechanisms through which phosphomimetic Dicer1 …

Tcf4 Is A Key Mediator Of Cell Identity And Oncogenesis In Neuroblastoma, Nour Aljouda

Theses and Dissertations (ETD)

Neuroblastomas (NB) are embryonal childhood tumors that derive from the multipotent neural crest cells (NCCs) of the peripheral nervous system. NB accounts for more than 15% of all childhood cancer-related deaths. Despite the most intensive multimodal therapy, more than 50% of patients with high-risk NB relapse with often fatal, resistant disease. Novel therapies are desperately needed to improve cure rates. Previous studies proposed that the deregulation of normal neural crest developmental programs contributes to NB oncogenesis by retaining the highly migratory and proliferative traits of NCCs. Thus, activation or repression of neural crest developmental pathways have been implicated in NB …

Examining Ndufab1 Expression In Head And Neck Squamous Cell Carcinoma, Addison Stevens

Poster Presentations

Honors research poster.

Introduction: Head and neck squamous cell carcinoma (HNSCC) is approximately 4% of all cancers and 2% of all cancer associated mortality in the United States. In 2023, there will be an estimated 67,000 new cases of HNSCC, along with 15,400 deaths, in the United States. HNSCC locations include the oral cavity, oropharynx, nasopharynx, hypopharynx, and larynx. Major risk factors for HNSCC include tobacco use, alcohol use, and human papilloma virus (HPV). Epidermal growth factor receptor (EGFR) is currently the only approved molecular targeted therapy for HNSCC. Therefore, new therapeutics and biomarkers for HNSCC are warranted. Mitochondria are …

Examining Ndufab1 Expression In Head And Neck Squamous Cell Carcinoma, Addison L. Stevens

Honors Theses

Head and neck squamous cell carcinoma (HNSCC) accounts for around 4% of all cancers in the USA. HNSCC includes cancers of the oral cavity, oropharynx, nasopharynx, hypopharynx, and larynx. Reprogramming of mitochondrial metabolism has been known to promote oncogenesis. NDUFAB1, a nuclear encoded subunit of respiratory complex I (RCI) in the inner mitochondrial membrane, is abundantly expressed at the mRNA level in HNSCC patients. Based on this finding, we hypothesize that NDUFAB1 protein expression is high in HNSCC and that NDUFAB1 expression predicts a poor prognosis in HNSCC patients. We determined NDUFAB1 expression in HNSCC using immunohistochemistry and pathology guided …

A Facile Strategy For The Fabrication Of Cell-Laden Porous Alginate Hydrogels Based On Two-Phase Aqueous Emulsions, Wen Xue, Donghee Lee, Yunfan Kong, Mitchell A. Kuss, Ying Huang, Taesung Kim, Soonkyu Chung, Andrew T. Dudley, Seung-Hyun Ro, Bin Duan

Journal Articles: Genetics, Cell Biology & Anatomy

Porous alginate (Alg) hydrogels possess many advantages as cell carriers. However, current pore generation methods require either complex or harsh fabrication processes, toxic components, or extra purification steps, limiting the feasibility and affecting the cellular survival and function. In this study, a simple and cell-friendly approach to generate highly porous cell-laden Alg hydrogels based on two-phase aqueous emulsions is reported. The pre-gel solutions, which contain two immiscible aqueous phases of Alg and caseinate (Cas), are cross-linked by calcium ions. The porous structure of the hydrogel construct is formed by subsequently removing the Cas phase from the ion-cross-linked Alg hydrogel. Those …

Molecular Subtyping And Survival Analysis Of Osteosarcoma Reveals Prognostic Biomarkers And Key Canonical Pathways, Siddesh Southekal, Sushil Kumar Shakyawar, Prachi Bajpai, Amr Elkholy, Upender Manne, Nitish Kumar Mishra, Chittibabu Guda

Journal Articles: Genetics, Cell Biology & Anatomy

Osteosarcoma (OS) is a common bone malignancy in children and adolescents. Although histological subtyping followed by improved OS treatment regimens have helped achieve favorable outcomes, a lack of understanding of the molecular subtypes remains a challenge to characterize its genetic heterogeneity and subsequently to identify diagnostic and prognostic biomarkers for developing effective treatments. In the present study, global analysis of DNA methylation, and mRNA and miRNA gene expression in OS patient samples were correlated with their clinical characteristics. The mucin family of genes, MUC6, MUC12, and MUC4, were found to be highly mutated in the OS patients. …

Rescue Of Auditory Function By A Single Administration Of Aav-Tmprss3 Gene Therapy In Aged Mice Of Human Recessive Deafness Dfnb8, Wan Du, Volkan Ergin, Corena Loeb, Mingqian Huang, Stewart Silver, Ariel Miura Armstrong, Zaohua Huang, Channabasavaiah B. Gurumurthy, Hinrich Staecker, Xuezhong Liu, Zheng-Yi Chen

Journal Articles: Genetics, Cell Biology & Anatomy

Patients with mutations in the TMPRSS3 gene suffer from recessive deafness DFNB8/DFNB10. For these patients, cochlear implantation is the only treatment option. Poor cochlear implantation outcomes are seen in some patients. To develop biological treatment for TMPRSS3 patients, we generated a knockin mouse model with a frequent human DFNB8 TMPRSS3 mutation. The Tmprss3^A306T/A306T homozygous mice display delayed onset progressive hearing loss similar to human DFNB8 patients. Using AAV2 as a vector to carry a human TMPRSS3 gene, AAV2-hTMPRSS3 injection in the adult knockin mouse inner ear results in TMPRSS3 expression in the hair cells and the spiral ganglion neurons. …

Molecular Mechanisms Of Prdm16 As A Tumor Suppressor In Pancreatic Ductal Adenocarcinoma, Eric Hurwitz

Theses and Dissertations

The transcription factor Prdm16 functions as a potent suppressor of transforming growth factor-beta (TGF-b) signaling, whose inactivation is deemed essential to the progression of pancreatic ductal adenocarcinoma (PDAC). Using the Kras^G12D-based mouse model of human PDAC, we surprisingly found that ablating Prdm16 did not block but instead accelerated PDAC formation and progression, suggesting that Prdm16 might function as a tumor suppressor in this malignancy. Subsequent genetic experiments showed that ablating Prdm16 along with Smad4 resulted in a shift from a well-differentiated and confined neoplasm to a highly aggressive and metastatic disease, which was associated with a striking deviation …

Editorial: Epigenetics And Molecular Genetics Of Rare Tumors, Mauro Tognon, George A. Calin, Luis Del Valle

School of Medicine Faculty Publications

No abstract provided.

Mistranslating Trnas Alter The Heat Shock Activation By Hsf1, Rebecca Dib

Undergraduate Student Research Internships Conference

Translation, or the production of protein from an mRNA blueprint, is among the most fundamental processes to life as we know it. tRNAs are essential to accurate translation, as they decode the codons of mRNA and recruit corresponding amino acids. Variant tRNAs with anticodon mutations can decrease translational fidelity by recruiting the incorrect amino acid, an aberrant process known as mistranslation. When proteins are produced with incorrect amino acid sequences, they may misfold. The heat shock response functions to alleviate cellular stress caused by misfolded proteins, either by refolding or targeting misfolded proteins for degradation. Hsf1 acts as a transcriptional …

Primary Cilia Of The Cardiac Neural Crest & Hedgehog-Mediated Mechanisms Of Congenital Heart Disease, Lindsey A. Fitzsimons

Electronic Theses and Dissertations

Elimination of primary cilia in cardiac neural crest cell (CNCC) progenitors is hypothesized to cause a variety of congenital heart defects (CHDs), including atrioventricular septal defects, and malformations of the developing cardiac outflow tract. We present an in vivo model of CHD resulting from the conditional elimination of primary cilia from CNCC using multiple, Wnt1:Cre-loxP, neural crest-specific systems, targeting two distinctive, but critical, primary cilia structural genes: Intraflagellar transport protein 88 (Ift88) or kinesin family member 3A (Kif3a). CNCC loss of primary cilia leads to widespread CHD, where homozygous mutant embryos (MUT) display a variety of outflow tract malformations, septation …

A Patient-Derived Ipsc Model To Study Glutamate Deficiency By Shank-3 Mutation In Autism Spectrum Disorder, Tiffany Berry, Courtney Caccia

Biology Student Scholarship

Tiffany Berry ’22, Majors: Biology and Psychology

Courtney Caccia ’22, Majors: Biology and Psychology

Faculty Mentor: Dr. Charles Toth, Biology

The use of human stem cell lines derived from persons with Autism Spectrum Disorder (ASD) provides a unique opportunity to model brain growth and potential to regain brain activity for treatment. Our lab has previously used stem cells to derive 3D cardiomyocytes to examine cardiovascular disease as well as kidney organoids and macrophages to study kidney disease. Using techniques our lab has learned using these stem cell models have prepared us to examine cell communication in mutated neurons. We will …

Anthracyclines Attenuate The Nrf1-Mediated Bounce-Back Response, Bader Albalawi

Theses and Dissertations

Proteasome inhibitors, such as carfilzomib, are FDA-approved to treat multiple myeloma and mantle cell lymphoma. Unfortunately, proteasome inhibitors have only produced clinically significant results in patients with hematologic cancers, despite their predicted pan-cancer utility, and even hematologic cancer types frequently show intrinsic and acquired resistance.

One proposed mechanism responsible for the proteasome inhibitors' shortcomings is the NRF1-mediated bounce-back response. Identification of drugs that can potentiate the action of proteasome inhibitors could overcome resistance in patients with hematologic cancers and expand proteasome inhibitors' use to treat solid tumors. Our previous studies have identified anthracyclines as potential compounds that interfere with the …

Extracellular Mechanotransduction, Stephen J. Haller, Andrew T. Dudley

Journal Articles: Genetics, Cell Biology & Anatomy

We highlight the force-sensing function of extracellular matrix and present a complementary mechanotransduction paradigm.

Mintruls: Prediction Of Mirna-Mrna Target Site Interactions Using Regularized Least Square Method, Sushil Kumar Shakyawar, Siddesh Southekal, Chittibabu Guda

Journal Articles: Genetics, Cell Biology & Anatomy

Identification of miRNA-mRNA interactions is critical to understand the new paradigms in gene regulation. Existing methods show suboptimal performance owing to inappropriate feature selection and limited integration of intuitive biological features of both miRNAs and mRNAs. The present regularized least square-based method, mintRULS, employs features of miRNAs and their target sites using pairwise similarity metrics based on free energy, sequence and repeat identities, and target site accessibility to predict miRNA-target site interactions. We hypothesized that miRNAs sharing similar structural and functional features are more likely to target the same mRNA, and conversely, mRNAs with similar features can be targeted by …

Identifying The Molecular Cause Of Extreme Endoplasmic Reticulum Dilation In Pediatric Osteosarcoma And Its Relationship To The Disease, Rachael Wood

Theses and Dissertations (ETD)

Pediatric osteosarcoma tumors are characterized by an unusual abundance of grossly dilated endoplasmic reticulum and an immense genomic instability that has complicated identifying new effective molecular therapeutic targets. Here we report a novel molecular signature that encompasses the majority of 108 patient tumor samples, PDXs and osteosarcoma cell lines. These tumors exhibit reduced expression of four critical COPII vesicle proteins that has resulted in the accumulation of procollagen-I protein within ‘hallmark’ dilated ER. Using CRISPR activation technology, increased expression of only SAR1A and SEC24D to physiologically normal levels was sufficient to restore both collagen-I secretion and resolve dilated ER morphology …

Role Of Smad2 And Smad3 On Adipose Tissue Development And Function, Roshan Kumari

Theses and Dissertations (ETD)

Introduction: Obesity and its associated metabolic syndrome are major medical problems worldwide including United States. Adipose tissue is the primary site of energy storage, playing important roles in health. Adipose tissue also has other critical functions, producing adipocytokines and contributing to normal nutrient metabolism, which in turn play important roles in satiety, inflammation, and total energy homeostasis. Activin A and activin B play important roles in maintaining body composition and energy homeostasis. This dissertation highlights the role of activin/SMADs signaling in adipose tissue development, function, and maintenance.

SMAD2/3 proteins are downstream mediators of transforming growth factor-β (TGFβ) family signaling, including …

Gene Expression Profiling Of Mapk Pathway Inhibitor Resistance In Cutaneous Melanoma: Can Bioinformatics Be Used To Select Better Melanoma Cell Lines?, Stephen Luebker

Theses & Dissertations

Melanoma is the deadliest form of skin cancer, and incidence has continued to increase. Half of all melanomas have a BRAF V600E mutation and respond to MAPK pathway inhibitors, including BRAF inhibitor therapy or BRAF/MEK inhibitor combination therapy, but nearly all patients develop treatment resistance. Melanoma cell lines produce variable results as models of MAPK pathway inhibitor resistance. To better understand how the genomic similarity of a melanoma cell line to patient-derived tumors affects resistance mechanisms, differences in DNA mutations and copy-number alterations were compared between melanoma cell lines profiled by the Cancer Cell Line Encyclopedia and cutaneous melanoma tumors …

Vascular Disease Pathogenesis In Smooth Muscle Dysfunction Syndrome And Majewski Osteodysplastic Primordial Dwarfism Type Ii, Jamie Wright

Dissertations & Theses (Open Access)

Vascular diseases are a leading cause of morbidity and mortality world-wide. Understanding their pathogenesis is crucial to better diagnosis and management of these life-threatening conditions. Through the study of rare mutations that lead to early onset and severe vascular diseases, we can elucidate underlying mechanisms for vascular disease pathogenesis and develop better treatments to prevent and manage more common causes of vascular diseases. In this study we look at two rare diseases that lead to severe vascular phenotypes, Smooth Muscle Dysfunction Syndrome (SMDS) and Majewski Osteodysplastic Primordial Dwarfism Type II (MOPDII). SMDS is a rare condition due to pathogenic variants …

Evaluation Of Somatic Mutations In Solid Metastatic Pan-Cancer Patients, Moom Roosan, Isa Mambetsariev, Rebecca Pharaon, Jeremy Fricke, Angel R. Baroz, Joseph Chao, Chen Chen, Mohd W. Nasser, Ramakanth Chirravuri-Venkata, Maneesh Jain, Lynette Smith, Susan E. Yost, Karen L. Reckamp, Raju Pillai, Leonidas Arvanitis, Michelle Afkhami, Edward W. Wang, Vincent Chung, Mihaela Cristea, Marwan Fakih, Marianna Koczywas, Erminia Massarelli, Joanne Mortimer, Yuan Yuan, Surinder K. Batra, Sumanta Pal, Ravi Salgia

Pharmacy Faculty Articles and Research

Metastasis continues to be the primary cause of all cancer-related deaths despite the recent advancements in cancer treatments. To evaluate the role of mutations in overall survival (OS) and treatment outcomes, we analyzed 957 metastatic patients with seven major cancer types who had available molecular testing results with a FoundationOne CDx^® panel. The most prevalent genes with somatic mutations were TP53, KRAS, APC, and LRP1B. In this analysis, these genes had mutation frequencies higher than in publicly available datasets. We identified that the somatic mutations were seven mutually exclusive gene pairs and an additional fifty-two co-occurring gene pairs. Mutations …

The Effects Of Mapk Signaling On The Development Of Cerebellar Granule Cells, Kerry Morgan

Honors Scholar Theses

The granule cells are the most abundant neuronal type in the human brain. Rapid proliferation of granule cell progenitors results in dramatic expansion and folding of the cerebellar cortex during postnatal development. Mis-regulation of this proliferation process causes medulloblastoma, the most prevalent childhood brain tumor. In the developing cerebellum, granule cells are derived from Atoh1-expressing cells, which arise from the upper rhombic lip (the interface between the roof plate and neuroepithelium). In addition to granule cells, the Atoh1 lineage also gives rise to different types of neurons including cerebellar nuclei neurons. In the current study, I have investigated the …

Ciliary Extracellular Vesicles Are Distinct From The Cytosolic Extracellular Vesicles, Ashraf M. Mohieldin, Rajasekharreddy Pala, Richard Beuttler, James J. Moresco, John R. Yates Iii, Surya M. Nauli

Pharmacy Faculty Articles and Research

Extracellular vesicles (EVs) are cell‐derived membrane vesicles that are released into the extracellular space. EVs encapsulate key proteins and mediate intercellular signalling pathways. Recently, primary cilia have been shown to release EVs under fluid‐shear flow, but many proteins encapsulated in these vesicles have never been identified. Primary cilia are ubiquitous mechanosensory organelles that protrude from the apical surface of almost all human cells. Primary cilia also serve as compartments for signalling pathways, and their defects have been associated with a wide range of human genetic diseases called ciliopathies. To better understand the mechanism of ciliopathies, it is imperative to know …

Trna Regulation In Humans: The Cellular Effect Of A Pathological Hars Y454s Mutation, Rosan Kenana

Electronic Thesis and Dissertation Repository

tRNAs are the adapter molecules involved in translating the genetic code into functional protein in a living cell. tRNAs are charged with their cognate amino acids - by aminoacyl-tRNA synthetases (aaRS or ARS) - which are then transferred to a growing peptide in a process called mRNA translation. The efficiency of translation is dependent on the ratio of ARS enzymes to their cognate tRNAs and the availability of correctly amino acylated tRNAs. Disruptions of this process, caused by mutations in ARS genes, in particular, have been linked to complex inherited diseases. USH3B syndrome, a recessively inherited disorder among consanguineous families …

Investigating The Role Of Znf384 Rearrangements In Acute Leukemia, Kirsten Dickerson

Theses and Dissertations (ETD)

Chromosomal rearrangements involving ZNF384 are the defining lesion in 5% of pediatric and adult B-cell acute lymphoblastic leukemia and tumors are characterized by aberrant myeloid marker expression. Additionally, ZNF384 rearrangements are the defining lesion in nearly half of pediatric B/myeloid mixed phenotype acute leukemia. These fusions juxtapose full-length ZNF384 to the N terminal portion of a diverse range of partners, most often, transcription factors or epigenetic modifiers. It has been shown that ZNF384-rearranged tumors have a distinct gene expression profile that is consistent between disease groups and N terminal partners. Genomic analyses of patient tumors has shown that ZNF384 fusions …

Fgdb: Database Of Follicle Stimulating Hormone Glycans, Sushil Kumar Shakyawar, Sanjit Pandey, David J. Harvey, George Bousfield, Chittibabu Guda

Journal Articles: Genetics, Cell Biology & Anatomy

Glycomics, the study of the entire complement of sugars of an organism has received significant attention in the recent past due to the advances made in high throughput mass spectrometry technologies. These analytical advancements have facilitated the characterization of glycans associated with the follicle-stimulating hormones (FSH), which play a central role in the human reproductive system both in males and females utilizing regulating gonadal (testicular and ovarian) functions. The irregularities in FSH activity are also directly linked with osteoporosis. The glycoanalytical studies have been tremendously helpful in understanding the biological roles of FSH. Subsequently, the increasing number of characterized FSH …