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Supplementary Tables 1-8 from Long-term In vitro Expansion Alters the Biology of Adult Mesenchymal Stem Cells

Supplementary Figure 1 from Long-term In vitro Expansion Alters the Biology of Adult Mesenchymal Stem Cells

Supplementary Figure 2C from Long-term In vitro Expansion Alters the Biology of Adult Mesenchymal Stem Cells

Mesenchymal stem cells (MSC) derived from bone marrow stem cells (BMSC) and adipose tissue stem cells (ASC) of humans and rhesus macaques were evaluated for their cell cycle properties during protracted culture in vitro. Human ASCs (hASC) and rhesus BMSCs (rBMSC) underwent significantly more total population doublings than human BMSCs (hBMSC) and rhesus ASCs (rASC). The cell cycle profile of all MSCs was altered as cultures aged. hMSCs underwent an increase in the frequency of cells in the S phase at P20 and P30. However, rhesus MSCs from both sources developed a distinct polyploid population of cells at P20, which progressed to aneuploidy by P30. Karyotype analysis of MSCs revealed the development of tetraploid or aneuploid karyotypes in the rhesus cells at P20 or P30. Analysis of the transcriptome of the MSCs from early and late passages revealed significant alterations in the patterns of gene expression (8.8% of the genes were differentially expressed in hBMSCs versus hASCs, and 5.5% in rBMSCs versus rASCs). Gene expression changes were much less evident within the same cell type as aging occurred (0.7% in hMSCs and 0.9% in rMSC). Gene ontology analysis showed that functions involved in protein catabolism and regulation of pol II transcription were overrepresented in rASCs, whereas the regulation of IκB/nuclear factor-κB cascade were overrepresented in hBMSCs. Functional analysis of genes that were differentially expressed in rASCs and hBMSCs revealed that pathways involved in cell cycle, cell cycle checkpoints, protein-ubiquitination, and apoptosis were altered. [Cancer Res 2008;68(11):4229–38]

Since 2018, we have evaluated the effectiveness of various teaching technologies for training young investigators on translational research in cancer health disparities. The Southeast Partnership for Improving Research and Training in Cancer Health Disparities (SPIRIT-CHD) unites Moffitt Cancer Center and the Louisiana State University Health Sciences Center. One of the main components of the SPIRIT-CHD is the Cancer Research Education Program (CREP) for training undergraduate and medical students from underrepresented backgrounds. The CREP utilizes a web-based didactic curriculum to engage students at both institutions in biobanking, precision medicine, and cancer health disparities topics. We report experiences from our cross-institutional cancer education program, specifically evaluating the cohorts’ satisfaction and learning gains using various communication technologies and instructional approaches. Trainees completed a survey with questions evaluating the curriculum and technology. Trainees reported satisfaction with the flipped classroom model (FCM) content and overall program (mean score = 3.2, SD = 0.79), and would recommend the program to peers. Yet, despite improved program delivery, trainees felt interaction between the two sites (mean score = 1.5, SD = 0.85) and engagement with faculty (mean score = 2.80, SD = 1.14) could be improved. The technology with the highest reported use was e-mail, with a mean score of 4.6 (SD = 0.52). LinkedIn and Twitter had the lowest frequency of use with mean scores at 1.90 (SD = 0.99) and 1.30 (SD = 1.34). Our study highlights the successes and challenges of remote learning using technology to increase interaction and engagement among trainees and faculty in a multi-site cancer research training program.

As literature indicates, historic racism and implicit bias throughout academia have been profound metrics leading to a lack of diversity, as related to people from underrepresented groups according to race and ethnicity, among biomedical sciences graduate students in U.S. universities. Recognizing such challenges, a team of biomedical scientists and inclusivity educators developed and implemented a pilot training program within an academic health sciences center as an initial step to educate faculty and staff regarding their roles in the promotion of an inclusive academic environment, receptive to all students, including underrepresented students. The 3-h workshop included didactic modules, videos, teaching modules, and active attendee participation. Faculty and staff were presented common terminology and ways to promote the development of an inclusive and diverse academic workforce. Compared with pre-workshop, post-workshop survey results indicated a statistically significant improvement in attendee knowledge of correctly identifying definitions of “implicit bias,” “status leveling,” “color-blind racial attitudes,” “tokenism,” and “failure to differentiate.” Additionally, by the end of the workshop, participants had a statistically significant increase in self-perceptions regarding the importance of improving diversity and recognizing biases and stereotypes in graduate education, knowing what to say when interacting with people from different cultures, and the ability to acknowledge bias when mentoring students from groups underrepresented in the biomedical field. This preliminary initiative was successful in the introduction of faculty and staff to the importance of fostering an inclusive academic environment and thereby developing a diverse workforce.

The COVID-19 pandemic shut down undergraduate research programs across the United States. A group of 23 colleges, universities, and research institutes hosted remote undergraduate research programs in the life sciences during Summer 2020. Given the unprecedented offering of remote programs, we carried out a study to describe and evaluate them. Using structured templates, we documented how programs were designed and implemented, including who participated. Through focus groups and surveys, we identified programmatic strengths and shortcomings as well as recommendations for improvements from students' perspectives. Strengths included the quality of mentorship, opportunities for learning and professional development, and a feeling of connection with a larger community. Weaknesses included limited cohort building, challenges with insufficient structure, and issues with technology. Although all programs had one or more activities related to diversity, equity, inclusion, and justice, these topics were largely absent from student reports even though programs coincided with a peak in national consciousness about racial inequities and structural racism. Our results provide evidence for designing remote Research Experiences for Undergraduates (REUs) that are experienced favorably by students. Our results also indicate that remote REUs are sufficiently positive to further investigate their affordances and constraints, including the potential to scale up offerings, with minimal concern about disenfranchising students.

In-person undergraduate research experiences (UREs) promote students’ integration into careers in life science research. In 2020, the COVID-19 pandemic prompted institutions hosting summer URE programs to offer them remotely, raising questions about whether undergraduates who participate in remote research can experience scientific integration. To address this, we investigated indicators of scientific integration for students who participated in remote life science URE programs in summer 2020. We found that these students experienced gains in their scientific self-efficacy and scientific identity similar to results reported for in-person UREs. We also found that these students perceived high benefits and low costs of doing research at the outset of their programs, and their perceptions did not change despite the remote circumstances. Yet, their perceptions differed by program, indicating that programs differentially affected students’ perceptions of the costs of doing research. Finally, we observed that students with prior research experience made greater gains in self-efficacy and identity, as well as in their perceptions of the alignment of their values with those of the scientific community, in comparison to students with no prior research experience. This finding suggests that additional programming may be needed for undergraduates with no prior experience to benefit from remote research.

The COVID-19 pandemic shut down undergraduate research programs across the U.S. Twenty-three sites offered remote undergraduate research programs in the life sciences during summer 2020. Given the unprecedented offering of remote research experiences, we carried out a study to describe and evaluate these programs. Using structured templates, we documented how programs were designed and implemented, including who participated. Through focus groups and surveys, we identified programmatic strengths and shortcomings as well as recommendations for improvements from the perspectives of participating students. Strengths included the quality of mentorship, opportunities for learning and professional development, and development of a sense of community. Weaknesses included limited cohort building, challenges with insufficient structure, and issues with technology. Although all programs had one or more activities related to diversity, equity, inclusion, and justice, these topics were largely absent from student reports even though programs coincided with a peak in national consciousness about racial inequities and structural racism. Our results provide evidence for designing remote REUs that are experienced favorably by students. Our results also indicate that remote REUs are sufficiently positive to further investigate their affordances and constraints, including the potential to scale up offerings, with minimal concern about disenfranchising students.

Many highly effective therapies used to treat cancer have been associated with irreversible development of detrimental neurological sequelae following the survival of childhood and adolescent cancer. While cancer treatment-induced late effects vary due to patient-related risk factors, cancer survivors who received chemotherapy or radiation may develop conductive or sensorineural hearing loss (SNHL). The purpose of this project is to determine which neuroinflammation-associated genes and biological pathways are affected by pediatric cancer treatments, and which treatment protocols are associated with increased ototoxicity. We hypothesize that cancer treatment causes abnormal gene expression of neuroinflammation and deafness-associated genes. The hypothesis will be tested with two specific aims. Aim I is to perform a retrospective review of diagnostic audiograms from pediatric cancer survivors enrolled in the Treatment After Cancer and Late Effects Center at Children’s Hospital New Orleans, Louisiana (CHNOLA). Aim II is to perform Nanostring neuroinflammation gene expression analysis followed by Ingenuity Pathway Analysis (IPA) of brain autopsy specimens from deceased pediatric patients who have previously undergone cancer treatment (Pathology Department, CHNOLA). Preliminary results revealed abnormal upregulation or downregulation of key genes, some of which are related to hearing loss and cochlear neuron degeneration when compared to age-matched controls (e.g., GJA1 and CASP3). The present study may provide information regarding which cancer treatment agents are ototoxic and reveal the candidate risk genes and pathways that contribute to auditory late effects. The long term goal of this project is to identify the needs of cancer survivors who are affected by treatment-induced hearing loss and provide them and their families with access to educational materials, medical resources, and social support to increase their health-related quality of life. Citation Format: Brittney T. Moore, Fern Tsien, Gabrielle Sheets, Ayesha Umrigar, Jordan Doss, Michael Norman, Matthew Stark, Pinki Prasad, Amanda Musso, Chindo Hicks, David Otohinoyi, Jovanny Zabaleta, Li Li. Ototoxicity of chemotherapy and radiation agents used in pediatric cancer treatment [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 3658.

Alveolar rhabdomyosarcoma (ARMS) is characterized by one of three translocation states: t(2;13)(q35;q14) producing PAX3-FOXO1, t(1;13)(p36;q14) producing PAX7-FOXO1, or translocation-negative. Tumors with t(2;13) are associated with greater disease severity and mortality than t(1;13) positive or translocation negative patients. We demonstrated that ARMS tumor samples contain intratumoral genetic heterogeneity with respect to the presence of t(2;13). Further, we demonstrated that this heterogeneity had an inverse correlation with tumor aggressiveness, with tumors containing higher translocation positivity having lower failure-free survival of patients. However, it is not yet known whether a correlation exists between percent t(2;13) positivity and changes in gene expression, in particular genes whose function contributes to immune invasion in the tumor microenvironment. Of fifty-five rhabdomyosarcoma patient tumor samples received from Children’s Hospital in New Orleans, nine were confirmed to contain t(2;13) ranging from 16 - 98% cells positive for the translocation. We used aged-matched normal muscle tissue derived from autopsies as a control. RNA was isolated from whole tissue samples and analyzed using the Nanostring nCounter®️ PanCancer IO 360™ Panel. This panel contains 770 genes that specifically analyze tumor immunogenicity, tumor sensitivity to immune attack, inhibitory immune mechanisms, stromal factors, inhibitory metabolism, anti-tumor immune activity, inhibitory immune signaling, immune cell population and abundance, and tumor inflammation. We found 487 genes have an average change in expression ≥ 1.5-fold relative to the control. Of these genes 31 (6.4%) have a statistically significant positive correlation and nearly 25% (114 genes) had a statistically significant negative correlation to percent intratumoral translocation positivity. A closer examination indicated that genes with a negative correlation had biological functions that target a variety of different immunological processes. These include the recognition of antigen by T cells; the activation, maturation, and differentiation of lymphocytes and myeloid cells; adhesion of immune responsive cells; promotion of the inflammatory response; and activation of the innate immune response. These results suggest that increased intratumoral t(2;13) positivity negatively impacts the immunological response thereby increasing the ability for immune evasion of the tumors with higher levels of the translocation, a result consistent with our previously published work. Citation Format: Andrew D. Hollenbach, Matthew Stark, Jovanny Zabaleta, Fern Tsien. Intratumoral translocation positive heterogeneity in pediatric alveolar rhabdomyosarcoma tumors correlates to changes in genes predicted to inhibit the immunological response [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 2355.

Since research shows that interactive prevention programs are more effective than didactic-based lectures, the current study examined the efficacy of an interactive substance abuse prevention program for adolescents. Participants completed pre- and post-surveys examining their substance use knowledge and program perceptions. The program included small groups led by near-peer health care professional trainees as facilitators and interactive, hands-on activities, such as the examination of diseased human organs due to substance use. Results demonstrated statistically significant increases in knowledge of substance use risks on mental and physical health. Findings highlight effective methods that can be implemented by health care professionals to increase awareness of the harmful effects of substance use.

Cytogenetic analysis is a conventional technique utilized worldwide to diagnose chromosome instability and may indicate the presence of a genetic disorder or malignancy; historically, it was one of the first techniques utilized for precision medicine. The field of clinical human cytogenetics (the study of human chromosomes, their structure, inheritance, and abnormalities) intensified with the discovery of the normal human chromosome number of 46 in 1956 and of trisomy 21 in Down syndrome in 1957. The presence of a 9; 22 translocation found in a patient with chronic myelogenous leukemia in 1959 led to the development of therapies targeting the oncogenic fusion protein produced by the “Philadelphia chromosome.” Today, cytogenetic analysis continues to provide essential diagnostic, prognostic, and therapeutic information for cancer precision medicine and research.

// Jacob M. Loupe 1,4,* , Patrick J. Miller 1,5,* , Benjamin P. Bonner 1,* , Elaine C. Maggi 1 , Jyothi Vijayaraghavan 1 , Jovanny Zabaleta 2 , Christopher M. Taylor 3 , Fern Tsien 1 , Judy S. Crabtree 1 and Andrew D. Hollenbach 1 1 Department of Genetics, Louisiana State University Health Sciences Center, New Orleans, LA, USA 2 Department of Pediatrics and Stanley S. Scott Cancer Center, Louisiana State University Health Sciences Center, New Orleans, LA, USA 3 Department of Microbiology, Immunology, and Parasitology, Louisiana State University Health Sciences Center, New Orleans, LA, USA 4 Center for Human Genetic Research, Massachusetts General Hospital, Richard B. Simches Research Center, Boston, MA, USA 5 Tulane University, New Orleans, LA, USA * These authors have contributed equally to this work Correspondence to: Andrew D. Hollenbach, email: // Keywords : aneuploidy, Pax3-FOXO1, myogenesis, phosphorylation, alveolar rhabdomyosarcoma Received : April 07, 2016 Accepted : August 10, 2016 Published : August 30, 2016 Abstract While many solid tumors are defined by the presence of a particular oncogene, the role that this oncogene plays in driving transformation through the acquisition of aneuploidy and overcoming growth arrest are often not known. Further, although aneuploidy is present in many solid tumors, it is not clear whether it is the cause or effect of malignant transformation. The childhood sarcoma, Alveolar Rhabdomyosarcoma (ARMS), is primarily defined by the t(2;13)(q35;q14) translocation, creating the PAX3-FOXO1 fusion protein. It is unclear what role PAX3-FOXO1 plays in the initial stages of tumor development through the acquisition and persistence of aneuploidy. In this study we demonstrate that PAX3-FOXO1 serves as a driver mutation to initiate a cascade of mRNA and miRNA changes that ultimately reprogram proliferating myoblasts to induce the formation of ARMS. We present evidence that cells containing PAX3-FOXO1 have changes in the expression of mRNA and miRNA essential for maintaining proper chromosome number and structure thereby promoting aneuploidy. Further, we demonstrate that the presence of PAX3-FOXO1 alters the expression of growth factor related mRNA and miRNA, thereby overriding aneuploid-dependent growth arrest. Finally, we present evidence that phosphorylation of PAX3-FOXO1 contributes to these changes. This is one of the first studies describing how an oncogene and post-translational modifications drive the development of a tumor through the acquisition and persistence of aneuploidy. This mechanism has implications for other solid tumors where large-scale genomics studies may elucidate how global alterations contribute to tumor phenotypes allowing the development of much needed multi-faceted tumor-specific therapeutic regimens.

The Southeast Partnership for Improving Research and Training in Cancer Health Disparities (SPIRIT-CHD) unites Louisiana State University Health Sciences Center (LSUHSC) and Moffitt Cancer Center (MCC) to advance translational research on cancer health disparities and to establish a Cancer Research Education Program (CREP). The CREP addresses a national priority to develop an educational training pipeline for one-on-one mentoring of undergraduates and medical students by a diverse group of LSUHSC and MCC faculty with unique expertise to conduct cancer health disparities research and outreach in underserved communities. The CREP supports 8-week internships providing: (1) hands-on summer research experiences; (2) a curriculum focusing on biobanking, precision medicine, and cancer health disparities; and (3) community outreach experiences in underserved communities. The curriculum includes web-based training modules, immersion experiences (e.g., biobank tour), professional development workshops, and learning activities (e.g., book and journal clubs). Data from the students' pre/post summative (impact/outcome) evaluations determine the acceptability and impact of these research and educational activities, students' knowledge, career aspirations, goal attainment, and their satisfaction based on nationally normed scales. Baseline and post-training data will be analyzed in August, at training completion, to assess program impact. Long-term yearly follow-up data will focus on the impact of CREP on student career trajectories. These data will help modify the CREP for years 2-4 of the SPIRIT-CHD. Seventy-five percent of the student participants in the first cohort were female. Students self-identified as Black/African American non-Hispanic (62.5%), White Hispanic (25%), and Asian non-Hispanic (12.5%). Student projects included genomic, immunologic, and cellular wet-lab research (analyzing proliferation of renal cell carcinoma, RNA sequencing and bioinformatics of Luminal B breast carcinomas, expression differences of polyamine enzymes in prostate cancer) and clinical studies (detection, prevention, and treatment of anal cancer in HIV-positive populations). Dry-lab projects focused on the analysis of Behavioral Risk Factor Surveillance System to assess policy-related trends in colorectal cancer screenings, studying the effectiveness of barbers as lay health educators for skin cancer prevention, and smoking cessation among Hispanics. CREP students also participated in cancer education outreach events to explain their projects to the communities at an elevated risk for certain types of cancer. This program has applicability to undergraduate and medical students nationwide on best practices for efficacious training in cancer health disparities, precision medicine, and biobanking. Ultimately, these efforts will enhance the diversity of the cancer research workforce, while contributing to the reduction of cancer health disparities. Citation Format: Fern Tsien, Paula Gregory, Gwendolyn Quinn, Vani N. Simmons, Z'Kera Sims, Megan E. Sutter, Ayesha Umrigar, Arnold H. Zea, Cathy Meade, Clement K. Gwede. The Cancer Research Education Program (CREP): Training the next generation of cancer health disparities researchers through the Southeast Partnership for Improving Research and Training in Cancer Health Disparities (SPIRIT-CHD) [abstract]. In: Proceedings of the Eleventh AACR Conference on the Science of Cancer Health Disparities in Racial/Ethnic Minorities and the Medically Underserved; 2018 Nov 2-5; New Orleans, LA. Philadelphia (PA): AACR; Cancer Epidemiol Biomarkers Prev 2020;29(6 Suppl):Abstract nr A065.

Triple-negative breast cancer (TNBC) represents a diverse group of breast cancers that are characterized by lack of expression of the estrogen receptor (ER), progesterone receptor (PR) and lack of amplification of the human epidermal growth factor (HER2) [1- 2].

Xeroderma pigmentosum (XP) is a rare autosomal recessive condition characterized by extreme sensitivity to ultraviolet light. Individuals with XP lack the ability to repair DNA (deoxyribonucleic acid) damage caused by ultraviolet radiation, leading to sunburn and increased susceptibility to skin cancers. Approximately 25% of patients also exhibit neural degeneration, which includes progressive mental deterioration, cortical thinning, and sensorineural hearing loss.Herein, we describe the audiological and genetic findings in a patient with XP subtype D with neural degeneration and hearing loss.This is a case report of a patient with XP subtype D, type 1 diabetes, and some clinical features typical of Charcot-Marie-Tooth disease.We obtained audiological evaluations over a course of 11 yr, including serial audiograms, auditory processing disorders evaluations, and electrophysiological testing.Hearing sensitivity has progressed from a unilateral mild high-frequency sensorineural hearing loss to a bilateral sloping moderate to severe/profound sensorineural hearing loss. In addition to the dramatic decline in hearing sensitivity, the patient demonstrates global auditory processing deficits, indicating a central component to his hearing loss.These findings emphasize the importance of the contribution of audiological evaluations to the diagnosis of a genetic disorder. Periodic evaluations of hearing sensitivity and auditory processing can provide information on disease progression in patients with XP with neural degeneration.

Mesenchymal stem cells (MSC) derived from bone marrow stem cells (BMSC) and adipose tissue stem cells (ASC) of humans and rhesus macaques were evaluated for their cell cycle properties during protracted culture in vitro. Human ASCs (hASC) and rhesus BMSCs (rBMSC) underwent significantly more total population doublings than human BMSCs (hBMSC) and rhesus ASCs (rASC). The cell cycle profile of all MSCs was altered as cultures aged. hMSCs underwent an increase in the frequency of cells in the S phase at P20 and P30. However, rhesus MSCs from both sources developed a distinct polyploid population of cells at P20, which progressed to aneuploidy by P30. Karyotype analysis of MSCs revealed the development of tetraploid or aneuploid karyotypes in the rhesus cells at P20 or P30. Analysis of the transcriptome of the MSCs from early and late passages revealed significant alterations in the patterns of gene expression (8.8% of the genes were differentially expressed in hBMSCs versus hASCs, and 5.5% in rBMSCs versus rASCs). Gene expression changes were much less evident within the same cell type as aging occurred (0.7% in hMSCs and 0.9% in rMSC). Gene ontology analysis showed that functions involved in protein catabolism and regulation of pol II transcription were overrepresented in rASCs, whereas the regulation of IκB/nuclear factor-κB cascade were overrepresented in hBMSCs. Functional analysis of genes that were differentially expressed in rASCs and hBMSCs revealed that pathways involved in cell cycle, cell cycle checkpoints, protein-ubiquitination, and apoptosis were altered. [Cancer Res 2008;68(11):4229–38]

We report on the phenotypic features of a patient with partial trisomy of the long arm of chromosome 16 due to an unbalanced Y;16 translocation (46,X,der[Y]t[Y;16] [q12;q22]pat). The patient was noted to have craniofacial anomalies and developmental delay, but no other major malformations. The father, a balanced Y;16 translocation carrier, has apparently normal fertility.

Untreated cultures from normal chorionic villus (CV) or amniotic fluid-derived (AF) samples displayed dramatic cell passage-dependent increases in aberrations in the juxtacentromeric heterochromatin of chromosomes 1 or 16 (1qh or 16qh). They showed negligible levels of chromosomal aberrations in primary culture and no other consistent chromosomal abnormality at any passage. By passage 8 or 9, 82 ± 7% of the CV metaphases from all eight studied samples exhibited 1qh or 16qh decondensation and 25 ± 16% had rearrangements in these regions. All six analyzed late-passage AF cultures displayed this regional decondensation and recombination in 54 ± 16 and 3 ± 3% of the metaphases, respectively. Late-passage skin fibroblasts did not show these aberrations. The chromosomal anomalies resembled those diagnostic for the ICF syndrome (immunodeficiency, centromeric region instability, and facial anomalies). ICF patients have constitutive hypomethylation at satellite 2 DNA (Sat2) in 1qh and 16qh, generally as the result of mutations in the DNA methyltransferase gene DNMT3B. At early and late passages, CV DNA was hypomethylated and AF DNA was hypermethylated both globally and at Sat2. DNMT1, DNMT3A, or DNMT3B RNA levels did not differ significantly between CV and AF cultures or late and early passages. The high degree of methylation of Sat2 in late-passage AF cells indicates that hypomethylation of this repeat is not necessary for 1qh decondensation. Sat2 hypomethylation may nonetheless favor 1qh and 16qh anomalies because CV cultures, with their Sat2 hypomethylation, displayed 1qh and 16qh decondensation and rearrangements at significantly lower passage numbers than did AF cultures. Also, CV cultures had much higher ratios of ICF-like rearrangements to heterochromatin decondensation in chromosomes 1 and 16. These cultures may serve as models to help elucidate the biological consequences of cancer-associated satellite DNA hypomethylation.

We report on a patient with mosaicism for monosomy 18, a chromosomal abnormality that has been reported only once previously. The patient had cleft lip and palate and mild behavioral and academic problems. His phenotype was milder in comparison with the previously reported patient by Khalifa et al. [1996: Clin Genet 49:318-320]. Further case reports of this chromosomal anomaly will be needed to determine a consistent phenotypic pattern to assist in management and genetic counseling.

Chromosome (cytogenetic) analysis is widely used for the detection of chromosome instability. When followed by G-banding and molecular techniques such as fluorescence in situ hybridization (FISH), this assay has the powerful ability to analyze individual cells for aberrations that involve gains or losses of portions of the genome and rearrangements involving one or more chromosomes. In humans, chromosome abnormalities occur in approximately 1 per 160 live births(1,2), 60-80% of all miscarriages(3,4), 10% of stillbirths(2,5), 13% of individuals with congenital heart disease(6), 3-6% of infertility cases(2), and in many patients with developmental delay and birth defects(7). Cytogenetic analysis of malignancy is routinely used by researchers and clinicians, as observations of clonal chromosomal abnormalities have been shown to have both diagnostic and prognostic significance(8,9). Chromosome isolation is invaluable for gene therapy and stem cell research of organisms including nonhuman primates and rodents(10-13). Chromosomes can be isolated from cells of live tissues, including blood lymphocytes, skin fibroblasts, amniocytes, placenta, bone marrow, and tumor specimens. Chromosomes are analyzed at the metaphase stage of mitosis, when they are most condensed and therefore more clearly visible. The first step of the chromosome isolation technique involves the disruption of the spindle fibers by incubation with Colcemid, to prevent the cells from proceeding to the subsequent anaphase stage. The cells are then treated with a hypotonic solution and preserved in their swollen state with Carnoy's fixative. The cells are then dropped on to slides and can then be utilized for a variety of procedures. G-banding involves trypsin treatment followed by staining with Giemsa to create characteristic light and dark bands. The same procedure to isolate chromosomes can be used for the preparation of cells for procedures such as fluorescence in situ hybridization (FISH), comparative genomic hybridization (CGH), and spectral karyotyping (SKY)(14,15).

Advances in sequencing technologies and increased understanding of the contribution of genetics to congenital sensorineural hearing loss have led to vastly improved outcomes for patients and their families. Next-generation sequencing and diagnostic panels have become increasingly reliable and less expensive for clinical use. Despite these developments, the diagnosis of genetic sensorineural hearing loss still presents challenges for healthcare providers. Inherited sensorineural hearing loss has high levels of genetic heterogeneity and variable expressivity. Additionally, syndromic hearing loss (hearing loss and additional clinical abnormalities) should be distinguished from non-syndromic (hearing loss is the only clinical symptom). Although the diagnosis of genetic sensorineural hearing loss can be challenging, the patient’s family history and ethnicity may provide critical information, as certain genetic mutations are more common in specific ethnic populations. The early identification of the cause of deafness can benefit patients and their families by estimating recurrence risks for future family planning and offering the proper interventions to improve their quality of life. Collaboration between pediatricians, audiologists, otolaryngologists, geneticists, and other specialists are essential in the diagnosis and management of patients with hearing disorders. An early diagnosis is vital for proper management and care, as some clinical manifestations of syndromic sensorineural hearing loss are not apparent at birth and have a delayed age of onset. We present a case of Usher syndrome (congenital deafness and childhood-onset blindness) illustrating the challenges encountered in the diagnosis and management of children presenting with congenital genetic sensorineural hearing loss, along with helpful resources for clinicians and families.

Hands-on, inquiry based learning, and one-on-one mentoring are the most effective methods of disseminating scientific information pertaining to cancer. The Science Youth Initiative (SYI) is a K-12 science education partnership between the Louisiana State University Health Sciences Center in New Orleans (LSUHSC-NO) and local schools. The workshops are coordinated with LSUHSC-NO faculty and school teachers to maximize the level of student learning and complement their classroom curriculum and academic level. Since 2009, the SYI has reached more than 2100 K-12 students to date, of which about 77% were middle and high school students. A diverse group of trainees from the LSUHSC-NO Schools of Medicine, Graduate Studies, Public Health, Allied Health Professions, and Nursing help educate the K-12 students and serve as role models. The benefit of having a variety of health care professionals with a range of expertise is that students receive cancer information from multiple perspectives. The program utilizes the book, The Immortal Life of Henrietta Lacks by Rebecca Skloot, which is part of the curriculum in many schools. During these workshops, middle and high school students are able to stain and examine HeLa cell chromosomes, thereby learning about chromatin instability commonly associated with cancer. Students also learn about DNA isolation, PCR, restriction digestion, and gel electrophoresis as they engage in workshop discussions on science history, bioethics, the human papilloma virus (HPV), and cervical cancer. Pre and post workshop surveys were developed to evaluate the participants’ learning and retention of the material covered, self-confidence in the academic material, awareness of science career options, and evaluation of the program. Results of the current academic year demonstrate: (1) a 51% increase in test scores based on the material covered (2) 59% of students were interested in participating in a medical or research related internship after the workshop (3) 54% of students were interested in working part-time in the medical sciences after the workshop. Participating schools included rural, urban, public/charter and private schools. These varied in socioeconomic and ethnic backgrounds: some which had almost 100% African American/Hispanic student body, while others had a diverse distribution of African American, Hispanic, Asian, and Caucasian students. The program's use of HeLa cells as a cancer genetics education tool has been successful in making health sciences and cancer research topics more interesting and accessible, motivating a variety of students to improve their grades, and promoting various health science related career opportunities. Citation Format: Ayesha Umrigar, Mary Moore, Fern Tsien. The use of HeLa cells as a cancer genetics education tool for middle and high schools. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 3463.

The der(1)t(1;19)(p12;p11) has not been previously reported in myelodysplastic syndrome (MDS). Fluorescence in situ hybridization (FISH) using chromosome 1- and chromosome 19-specific probes, performed on the bone marrow (BM) cells of this patient confirmed the initial karyotype, i.e., 47,XY,+der(1) t(1;19)(p12;p11).

The isolation and characterization of embryonic and adult stem cells from higher-order mammalian species will enhance the understanding of the biology and therapeutic application of stem cells. The aim of this study was to purify rhesus mesenchymal stem cells (MSCs) from adult bone marrow and to characterize functionally their abilities to differentiate along diverse lineages. Adherent cells from adult rhesus macaque bone marrow were characterized for their growth characteristics, lineage differentiation, cell-surface antigen expression, telomere length, chromosome content, and transcription factor gene expression. Rhesus bone marrow MSCs (BMSCs) are very heterogeneous, composed of primarily long, thin cells and some smaller, round cells. The cells are capable of differentiating along osteogenic, chondrogenic, and adipogenic lineages in vitro. The cell morphology and multipotential differentiation capabilities are maintained throughout extended culture. They express CD59, CD90 (Thy-1), CD105, and HLA-1 and were negative for hematopoietic markers such as CD3, CD4, CD8, CD11b, CD13, CD34, and platelet endothelial cell adhesion molecule-1 (CD31). BMSCs were also demonstrated to express the mRNA for important stem cell-related transcription factors such as Oct-4, Sox-2, Rex-1, and Nanog. Rhesus BMSCs have a normal chromosome content, and the shortening of telomeres is minimal during early passages. These data demonstrate that BMSCs isolated from rhesus macaques have a high degree of commonality with MSCs isolated from other species. Therefore, isolation of these cells provides an effective and convenient method for rapid expansion of pluripotent rhesus MSCs.

Editor—The immunodeficiency, centromeric region instability, and facial anomalies syndrome (ICF) usually involves mutations affecting the catalytic domain in DNMT3B, one of the three human genes known to encode DNA methyltransferases.1-3 ICF always results in defective immunity, a high frequency of chromosomal abnormalities in the vicinity of the centromere (pericentromeric region) of chromosome 1 and/or chromosome 16 in mitogen stimulated lymphocytes, and hypomethylation of a small portion of the genome.4-6 ICF symptoms are manifested often from infancy and this syndrome can cause early childhood death from infections. The DNA sequences targeted for undermethylation in ICF include the heterochromatin adjacent to the centromeres of chromosomes 1 and 16 (1qh and 16qh), where a high incidence of chromatin decondensation, chromosome and chromatid breaks, and rearrangements to form multiradial chromosomes are characteristically seen in mitogen stimulated ICF blood cultures and in ICF lymphoblastoid cell lines.6-9 These aberrations are more common in chromosome 1 than in chromosome 16 and only infrequently observed in chromosome 9.5 8 9 We describe an unusual primary culture from a chorionic villus (CV) biopsy in which a high frequency of ICF-like chromosomal abnormalities was observed. However, follow up indicated that the infant did not have ICF. A 30 year old, gravida 4, para 0, ab 3 white female was referred for genetic counselling and CV sampling because of a previous pregnancy with trisomy 13. Both the patient and her husband were phenotypically normal and healthy apart from their reproductive history. The patient's first two pregnancies ended in spontaneous abortion at 15 and 10 weeks. No fetal studies had been performed; parental chromosomes were analysed and reported to be normal. The third pregnancy was found to be affected with trisomy 13 (47,XY,+13) and was then terminated. During the fourth pregnancy, the patient was offered and …

Item does not contain fulltext ICF (immunodeficiency, centromeric region instability and facial anomalies) is a recessive disease caused by mutations in the DNA methyltransferase 3B gene (DNMT3B). Patients have immunodeficiency, chromosome 1 (Chr1) and Chr16 pericentromeric anomalies in mitogen-stimulated lymphocytes, a small decrease in overall genomic 5-methylcytosine levels and much hypomethylation of Chr1 and Chr16 juxtacentromeric heterochromatin. Microarray expression analysis was done on B-cell lymphoblastoid cell lines (LCLs) from ICF patients with diverse DNMT3B mutations and on control LCLs using oligonucleotide arrays for approximately 5600 different genes, 510 of which showed a lymphoid lineage-restricted expression pattern among several different lineages tested. A set of 32 genes had consistent and significant ICF-specific changes in RNA levels. Half of these genes play a role in immune function. ICF-specific increases in immunoglobulin (Ig) heavy constant mu and delta RNA and cell surface IgM and IgD and decreases in Ig(gamma) and Ig(alpha) RNA and surface IgG and IgA indicate inhibition of the later steps of lymphocyte maturation. ICF-specific increases were seen in RNA for RGS1, a B-cell specific inhibitor of G-protein signaling implicated in negative regulation of B-cell migration, and in RNA for the pro-apoptotic protein kinase C eta gene. ICF-associated decreases were observed in RNAs encoding proteins involved in activation, migration or survival of lymphoid cells, namely, transcription factor negative regulator ID3, the enhancer-binding MEF2C, the iron regulatory transferrin receptor, integrin beta7, the stress protein heme oxygenase and the lymphocyte-specific tumor necrosis factor receptor family members 7 and 17. No differences in promoter methylation were seen between ICF and normal LCLs for three ICF upregulated genes and one downregulated gene by a quantitative methylation assay [combined bisulfite restriction analysis (COBRA)]. Our data suggest that DNMT3B mutations in the ICF syndrome cause lymphogenesis-associated gene dysregulation by indirect effects on gene expression that interfere with normal lymphocyte signaling, maturation and migration.

Facioscapulohumeral muscular dystrophy (FSHD) has an unusual molecular etiology. In a putatively heterochromatic subtelomeric region of each chromosome 4 homologue (4q35), unaffected individuals have 11 to about 95 tandem copies of a complex 3.3-kb repeat (D4Z4). Most FSHD patients have less than 10 copies at one allelic 4q35. This has been proposed to lead to the loss of heterochromatinization and, thereby, inappropriate gene expression by position effects, explaining the dominant nature of FSHD and the role of a decreased number of copies of D4Z4 at 4q35 but not at 10q26. Consistent with the proposed heterochromatinization of this repeat, by Southern blot analysis, we found that SmaI, MluI, SacII, and EagI sites in D4Z4 are highly methylated in normal and FSHD cell lines and somatic tissues, including skeletal muscle. Like repeated DNA sequences in the juxtacentromeric heterochromatin of chromosomes 1, 9, and 16, D4Z4 was hypomethylated at numerous CpGs in sperm and in cell lines from patients with an unrelated DNA methyltransferase deficiency syndrome (ICF; immunodeficiency, centromeric region instability, facial anomalies) in contrast to its hypermethylation in non-ICF postnatal somatic tissues. Our data on FSHD samples suggest that the disease-associated 4q35 D4Z4 repeats, which constitute a small percentage of the total D4Z4 repeats, are not generally hypomethylated relative to the other repeats of this sequence. However, in individuals not affected with FSHD, the hypermethylation of tandem, high-copy-number D4Z4 repeats might help stabilize heterochromatinization at allelic 4q35 regions just as hypermethylation elsewhere in the genome has been linked to chromatin compaction.

IL-7 is essential for T cell survival and development. IL-7 receptor (IL-7Rα) efficiency manifests SCID due to substantial thymocyte apoptosis. We hypothesized that IL-7Rαnull-induced apoptosis is partially mediated by an elevated p53 activity. To investigate the genetic association of IL-7Rα signaling with the p53 pathway, we generated IL-7Rαnullp53null (DKO) mice. DKO mice exhibited a marked reduction of apoptosis in developing T cells and an augmented thymic lymphomagenesis with telomere erosions and exacerbated chromosomal anomalies, including chromosome duplications, breaks, and translocations. In particular, Robertsonian translocations, in which telocentric chromosomes fuse at the centromeric region, and a complete loss of telomeres at the fusion site occurred frequently in DKO thymic lymphomas. Cellular and molecular investigations revealed that IL-7/IL-7Rα signaling withdrawal diminished the protein synthesis of POT1 (protection of telomere 1), a subunit of telomere protective complex shelterin, leading to telomere erosion and the activation of the p53 pathway. Blockade of IL-7/IL-7Rα signaling in IL-7-dependent p53null cells reduced POT1 expression and caused telomere and chromosome abnormalities similar to those observed in DKO lymphomas. This study underscores a novel function of IL-7/IL-7Rα during T cell development in regulating telomere integrity via POT1 expression and provides new insights into cytokine-mediated survival signals and T cell lymphomagenesis.

The objective of this study was to determine if measurement of initial crown--rump length (CRL) is helpful in predicting low birth weight, newborn length, spontaneous abortions, or abortus karyotype. We measured CRL prospectively in 837 consecutive singleton pregnancies at the time a heart rate was first detectable with transvaginal ultrasonography and compared these measurements to normal values for the 10th through 90th centiles determined from 227 transvaginal ultrasound measurements in in-vitro fertilization and gamete intra-Fallopian transfer pregnancies with known ovulation dates. The relationship of initial CRL to birth weight and length and to abortion and abortus karyotype was analysed after all pregnancies had delivered. Initial CRL measured after the 28th post-ovulation day was predictive of subsequent abortion, but not of low birth weight or length. The abortion rate was 3.3% [95% confidence interval (CI) 1.5%, 5.1%] when initial CRL > or = 50th centile, compared to 19.4% (95% CI 15.4%, 23.4%) when < 50th centile. Initial CRL was < 50th centile in 13 out of 14 trisomic and in eight out of 10 other karyotypically abnormal aborti. These results indicate that initial CRL measured after the 28th post-ovulation day may help to identify pregnancies at increased risk of abortion due to abnormal karyotypes.