Bioinformatics characterization of variants of uncertain significance in pediatric sensorineural hearing loss.

Introduction: Rapid advancements in Next Generation Sequencing (NGS) and bioinformatics tools have allowed physicians to obtain genetic testing results in a more rapid, cost-effective, and comprehensive manner than ever before. Around 50% of pediatric sensorineural hearing loss (SNHL) cases are due to a genetic etiology, thus physicians regularly utilize targeted sequencing panels that identify variants in genes related to SNHL. These panels allow for early detection of pathogenic variants which allows physicians to provide anticipatory guidance to families. Molecular testing does not always reveal a clear etiology due to the presence of multigenic variants with varying classifications, including the presence of Variants of Uncertain Significance (VUS). This study aims to perform a preliminary bioinformatics characterization of patients with variants associated with Type II Usher Syndrome in the presence of other multigenic variants. We also provide an interpretation algorithm for physicians reviewing molecular results with medical geneticists. Methods: Review of records for multigenic and/or VUS results identified several potential subjects of interest. For the purposes of this study, two ADGRV1 compound heterozygotes met inclusion criteria. Sequencing, data processing, and variant calling (the process by which variants are identified from sequence data) was performed at Invitae (San Francisco CA). The preliminary analysis followed the recommendations outlined by the American College of Medical Genetics and Association for Molecular Pathology (ACMG-AMP) in 2015 and 2019. The present study utilizes computational analysis, predictive data, and population data as well as clinical information from chart review and publicly available information in the ClinVar database. Results: Two subjects were identified as compound heterozygotes for variants in the gene ADGRV1. Subject 1's variants were predicted as deleterious, while Subject 2's variants were predicted as non-deleterious. These results were based on known information of the variants from ClinVar, multiple lines of computational data, population databases, as well as the clinical presentation. Discussion: Early molecular diagnosis through NGS is ideal, as families are then able to access a wide range of resources that will ultimately support the child as their condition progresses. We recommend that physicians build strong relationships with medical geneticists and carefully review their interpretation before making recommendations to families, particularly when addressing the VUS. Reclassification efforts of VUS are supported by studies like ours that provide evidence of pathogenic or benign effects of variants.

Is Methotrexate Ototoxic? Investigating the Ototoxic Late Effects of Pediatric Cancer Treatment.

PURPOSE: Pediatric cancer survivors often experience long-term adverse health conditions or late effects, including hearing loss, that are attributable to cancer therapy. Ototoxic late effects have been documented in patients with cancer treated with cisplatin-based chemotherapy and/or radiation. This study evaluated the late effects of methotrexate as compared to cisplatin and other cancer therapy agents on pediatric cancer survivors at the Children's Hospital of New Orleans in Louisiana (CHNOLA) and patients currently undergoing cancer treatment at Our Lady of the Lake (OLOL) Hospital in Baton Rouge, Louisiana. METHOD: A retrospective chart review was conducted of medical records from the CHNOLA Audiology Clinic and the Treatment After Cancer Late Effects clinic, which followed patients 2-19 years after cancer treatment completion and current patients with pediatric cancer at OLOL. This study identified pediatric cancer survivors between 2 and 24 years of age with treatment protocol information and audiological evaluations. Association studies were performed to calculate p values using an exact chi-square test. RESULTS: More than 44% of late-effects patients had significant hearing loss; mild-to-profound hearing loss was observed in 37.5% of patients who received methotrexate treatment without cisplatin or irradiation. Eighty-three percent of the patients who received cisplatin had late-effect hearing loss. In patients currently receiving cancer treatment, 12% had significant hearing loss. CONCLUSIONS: The results from this study suggest that children who receive therapies not clinically established as ototoxic (i.e., methotrexate) may still be at a high risk of developing long-term hearing loss as a late effect. Due to the high incidence rate of hearing loss among patients with pediatric cancer, we recommend that audiologists be part of the late-effects care team. This study also demonstrates that patients with pediatric cancer treated with methotrexate should receive routine long-term auditory monitoring as part of their standard of care to detect and manage hearing loss early, minimizing adverse outcomes.

Virtually the Same? Evaluating the Effectiveness of Remote Undergraduate Research Experiences.

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 and whether they might perceive doing research less favorably (i.e., not beneficial or too costly). To address these questions, we examined indicators of scientific integration and perceptions of the benefits and costs of doing research among students who participated in remote life science URE programs in Summer 2020. We found that students experienced gains in scientific self-efficacy pre- to post-URE, similar to results reported for in-person UREs. We also found that students experienced gains in scientific identity, graduate and career intentions, and perceptions of the benefits of doing research only if they started their remote UREs at lower levels on these variables. Collectively, students did not change in their perceptions of the costs of doing research despite the challenges of working remotely. Yet students who started with low cost perceptions increased in these perceptions. These findings indicate that remote UREs can support students' self-efficacy development, but may otherwise be limited in their potential to promote scientific integration.

"How Do We Do This at a Distance?!" A Descriptive Study of Remote Undergraduate Research Programs during COVID-19.

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.

Improving Multi-site Interaction Through Remote Learning Technology: Report from a Training Program to Increase Underrepresented Undergraduate and Medical Students in Health Disparities Research.

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.

Intratumoral Translocation Positive Heterogeneity in Pediatric Alveolar Rhabdomyosarcoma Tumors Correlates to Patient Survival Prognosis.

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. Consistent with this fact, previous work concluded that a molecular analysis of RMS translocation status is essential for the accurate determination of prognosis and diagnosis. However, despite this knowledge, most diagnoses rely on histology and in some cases utilize fluorescence in situ hybridization (FISH) probes unable to differentiate between translocation products. Along these same lines, diagnostic RT-PCR analysis, which can differentiate translocation status, is unable to determine intratumoral translocation heterogeneity, making it difficult to determine if heterogeneity exists and whether correlations exist between this heterogeneity and patient outcomes. Using newly developed FISH probes, we demonstrate that intratumoral heterogeneity exists in ARMS tumors with respect to the presence or absence of the translocation product. We found between 3 and 98% of cells within individual tumor samples contained a translocation event with a significant inverse correlation (R 2 = 0.66, p = 0.001) between the extent of intratumoral translocation heterogeneity and failure-free survival of patients. Taken together, these results provide additional support for the inclusion of the molecular analysis of these tumors and expand on this idea to support determining the extent of intratumoral translocation heterogeneity in the diagnosis of ARMS to improve diagnostic and prognostic indicators for patients with these tumors.

Knowledge gains in a professional development workshop on diversity, equity, inclusion, and implicit bias in academia.

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.

An Integrative Genomics Approach for Associating Genetic Susceptibility with the Tumor Immune Microenvironment in Triple Negative Breast Cancer.

Background: Triple-negative breast cancer (TNBC) is the most aggressive form of breast cancer. It is a heterogeneous disease with poor prognosis. Contributing to the worse prognosis in TNBC is the higher rates of relapse and rapid progression to metastatic disease which is often lethal. With the exception of cytotoxic chemotherapy, there is currently no effective targeted therapies. Immunotherapy such as vaccines offer new opportunities for treatment of TNBC. But realizing the potential of immunotherapy and vaccination may require understanding the association between the tumor immune microenvironment and genetic susceptibility to TNBC. The objective of this exploratory study was to investigate the potential association between genetic susceptibility and tumor immune microenvironment in TNBC. Methods: We integrated information on genetic variants and genes associated with an increased risk of developing breast cancer with gene expression data from the Caucasian women diagnosed with the basal-like immune activated (N=54) and basal-like immune suppressed (N=60) subtypes of TNBC to discover and characterize immune modulated gene signatures, molecular networks and biological pathways enriched for genetic susceptibility variants. Results: The investigation revealed immune modulated gene signatures, molecular networks and biological pathways enriched for genetic susceptibility variants. The discovered pathways included the role of BRCA1 in DNA damage response, hereditary breast cancer, aryl hydrocarbon receptor and molecular mechanisms of cancer signaling pathways. Conclusion: The investigation suggests the link between genetic susceptibility and the tumor immune microenvironment in TNBC and establishes putative functional bridges between genetic predisposition and immune modulated pathways.

Delayed diagnosis of a patient with Usher syndrome 1C in a Louisiana Acadian family highlights the necessity of timely genetic testing for the diagnosis and management of congenital hearing loss.

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.

Detailed Audiological Evaluation of a Patient with Xeroderma Pigmentosum with Neural Degeneration.

BACKGROUND: 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. PURPOSE: Herein, we describe the audiological and genetic findings in a patient with XP subtype D with neural degeneration and hearing loss. RESEARCH DESIGN: 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. DATA COLLECTION AND ANALYSIS: We obtained audiological evaluations over a course of 11 yr, including serial audiograms, auditory processing disorders evaluations, and electrophysiological testing. RESULTS: 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. CONCLUSION: 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.

Acquisition of an oncogenic fusion protein serves as an initial driving mutation by inducing aneuploidy and overriding proliferative defects.

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.

Chromosome preparation from cultured cells.

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).

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 I kappa B/nuclear factor-kappaB 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.

Characterization of multipotent mesenchymal stem cells from the bone marrow of rhesus macaques.

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.

Phenotypic features of a boy with trisomy of 16q22-->qter due to paternal Y; 16 translocation.

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.