Population-Based Prevalence of CDKN2A Mutations in Utah Melanoma Families

Cyclin-dependent kinase inhibitor 2A (CDKN2A or p16) is the major melanoma predisposition gene. In order to evaluate the candidacy for genetic testing of CDKN2A mutations among melanoma prone families, it is important to identify characteristics that predict a high likelihood of carrying a CDKN2A mutation. We primarily used a unique Utah genealogical resource to identify independent melanoma prone families whom we tested for mutations in CDKN2A, cyclin-dependent kinase 4, and alternate reading frame. We sampled 60 families which met the inclusion criteria of two or more affected first-degree relatives. We found four different pathogenic CDKN2A mutations in five families, mutations of uncertain significance in two families, and known polymorphisms in three families. One of the mutations of uncertain significance, 5′ untranslated region −25C>T, has not been previously described. Among our population-based set of Utah families, the prevalence of CDKN2A mutations was 8.2% (4/49); the overall prevalence when physician-referred pedigrees were also considered was between 8.3% (5/60) and 10% (6/60). Having four or more first- or second-degree relatives with melanoma, or a family member with ≥3 primary melanomas, correlated strongly with carrying a CDKN2A mutation. We observed a significantly elevated rate of pancreatic cancer in one of four families with a deleterious CDKN2A mutation

Cellular and ultrastructural characterization of the grey-morph phenotype in southern right whales (Eubalaena australis)

Southern right whales (SRWs, Eubalena australis) are polymorphic for an X-linked pigmentation pattern known as grey morphism. Most SRWs have completely black skin with white patches on their bellies and occasionally on their backs; these patches remain white as the whale ages. Grey morphs (previously referred to as partial albinos) appear mostly white at birth, with a splattering of rounded black marks; but as the whales age, the white skin gradually changes to a brownish grey color. The cellular and developmental bases of grey morphism are not understood. Here we describe cellular and ultrastructural features of grey-morph skin in relation to that of normal, wild-type skin. Melanocytes were identified histologically and counted, and melanosomes were measured using transmission electron microscopy. Grey-morph skin had fewer melanocytes when compared to wild-type skin, suggesting reduced melanocyte survival, migration, or proliferation in these whales. Grey-morph melanocytes had smaller melanosomes relative to wild-type skin, normal transport of melanosomes to surrounding keratinocytes, and normal localization of melanin granules above the keratinocyte nuclei. These findings indicate that SRW grey-morph pigmentation patterns are caused by reduced numbers of melanocytes in the skin, as well as by reduced amounts of melanin production and/or reduced sizes of mature melanosomes. Grey morphism is distinct from piebaldism and albinism found in other species, which are genetic pigmentation conditions resulting from the local absence of melanocytes, or the inability to synthesize melanin, respectively

Multiple primary melanomas in a CDKN2A mutation carrier exposed to ionizing radiation

Background: Recent research has shown a possible causal relationship between ionizing radiation exposure and melanoma. Individuals with mutations in CDKN2A (cyclin-dependent kinase inhibitor 2A), the major melanoma predisposition gene, have an increased susceptibility to melanoma-promoting exposures, such as UV light. We describe a patient from a familial melanoma pedigree with 7 primary melanomas on the right side of her body, the first occurring 5 years after exposure to atmospheric nuclear bomb testing in the 1950s. Observations: Physical examination revealed phototype I skin, red hair, and 26 nevi (14 on the right and 12 on the left side of her body). One nevus was larger than 5 mm, and 2 were clinically atypical. Sequence analysis demonstrated a known deleterious mutation in CDKN2A (G-34T) and homozygosity for a red hair color variant in MC1R (melanocortin 1 receptor) (R151C). Fluorescence in situ hybridization analysis of blood, fibroblasts, and melanocytes from both upper extremities ruled out mosaicism. Conclusions: Individuals such as this patient, who has CDKN2A and MC1R mutations, are likely to be more susceptible to environmental insults. A careful review of environmental exposures in these vulnerable cases may reveal cancer-promoting agents, such as ionizing radiation, that go unnoticed in less susceptible populations

Nucleic acid extraction from formalin-fixed paraffin-embedded cancer cell line samples: a trade off between quantity and quality?

Background: Advanced genomic techniques such as Next-Generation-Sequencing (NGS) and gene expression profiling, including NanoString, are vital for the development of personalised medicines, as they enable molecular disease classification. This has become increasingly important in the treatment of cancer, aiding patient selection. However, it requires efficient nucleic acid extraction often from formalin-fixed paraffin-embedded tissue (FFPE). Methods: Here we provide a comparison of several commercially available manual and automated methods for DNA and/or RNA extraction from FFPE cancer cell line samples from Qiagen, life Technologies and Promega. Differing extraction geometric mean yields were evaluated across each of the kits tested, assessing dual DNA/RNA extraction vs. specialised single extraction, manual silica column based extraction techniques vs. automated magnetic bead based methods along with a comparison of subsequent nucleic acid purity methods, providing a full evaluation of nucleic acids isolated. Results: Out of the four RNA extraction kits evaluated the RNeasy FFPE kit, from Qiagen, gave superior geometric mean yields, whilst the Maxwell 16 automated method, from Promega, yielded the highest quality RNA by quantitative real time RT-PCR. Of the DNA extraction kits evaluated the PicoPure DNA kit, from Life Technologies, isolated 2–14× more DNA. A miniaturised qPCR assay was developed for DNA quantification and quality assessment. Conclusions: Careful consideration of an extraction kit is necessary dependent on quality or quantity of material required. Here we provide a flow diagram on the factors to consider when choosing an extraction kit as well as how to accurately quantify and QC the extracted material

Impact of RNA degradation on gene expression profiling

<p>Abstract</p> <p>Background</p> <p>Gene expression profiling is a highly sensitive technique which is used for profiling tumor samples for medical prognosis. RNA quality and degradation influence the analysis results of gene expression profiles. The impact of this influence on the profiles and its medical impact is not fully understood. As patient samples are very valuable for clinical studies, it is necessary to establish criteria for the RNA quality to be able to use these samples in later analysis.</p> <p>Methods</p> <p>To investigate the effects of RNA integrity on gene expression profiling, whole genome expression arrays were used. We used tumor biopsies from patients diagnosed with locally advanced rectal cancer. To simulate degradation, the isolated total RNA of all patients was subjected to heat-induced degradation in a time-dependent manner. Expression profiling was then performed and data were analyzed bioinformatically to assess the differences.</p> <p>Results</p> <p>The differences introduced by RNA degradation were largely outweighed by the biological differences between the patients. Only a relatively small number of probes (275 out of 41,000) show a significant effect due to degradation. The genes that show the strongest effect due to RNA degradation were, especially, those with short mRNAs and probe positions near the 5' end.</p> <p>Conclusions</p> <p>Degraded RNA from tumor samples (RIN > 5) can still be used to perform gene expression analysis. A much higher biological variance between patients is observed compared to the effect that is imposed by degradation of RNA. Nevertheless there are genes, very short ones and those with the probe binding side close to the 5' end that should be excluded from gene expression analysis when working with degraded RNA. These results are limited to the Agilent 44 k microarray platform and should be carefully interpreted when transferring to other settings.</p

Circadian Rhythm and Cartilage Extracellular Matrix Genes in Osseointegration: A Genome-Wide Screening of Implant Failure by Vitamin D Deficiency

Successful dental and orthopedic implants require the establishment of an intimate association with bone tissue; however, the mechanistic explanation of how biological systems accomplish osseointegration is still incomplete. We sought to identify critical gene networks involved in osseointegration by exploring the implant failure model under vitamin D deficiency.Adult male Sprague-Dawley rats were exposed to control or vitamin D-deficient diet prior to the osteotomy surgery in the femur bone and the placement of T-shaped Ti4Al6V implant. Two weeks after the osteotomy and implant placement, tissue formed at the osteotomy site or in the hollow chamber of T-shaped implant was harvested and total RNA was evaluated by whole genome microarray analyses.Two-way ANOVA of microarray data identified 103 genes that were significantly (>2 fold) modulated by the implant placement and vitamin D deficiency. Kyoto Encyclopedia of Genes and Genomes (KEGG) analyses assigned the highest z-score to the circadian rhythm pathway including neuronal PAS domain 2 (NPAS2), and period homolog 2 (Per2). NPAS2 and Aryl hydrocarbon receptor nuclear translocator-like (ARNTL/Bmal 1) were upregulated around implant and diminished by vitamin D deficiency, whereas the expression pattern of Per2 was complementary. Hierarchical cluster analysis further revealed that NPAS2 was in a group predominantly composed of cartilage extracellular matrix (ECM) genes. Whereas the expression of bone ECM genes around implant was not significantly affected by vitamin D deficiency, cartilage ECM genes were modulated by the presence of the implant and vitamin D status. In a proof-of-concept in vitro study, the expression of cartilage type II and X collagens was found upregulated when mouse mesenchymal stem cells were cultured on implant disk with 1,25D supplementation.This study suggests that the circadian rhythm system and cartilage extracellular matrix may be involved in the establishment of osseointegration under vitamin D regulation

EC68-540 Nebraska Home Extension Club Membership

Extension Circular 68-540: Nebraska Home Extension Club Membership; An eighteen county survey

Personality Factors that Predict Cyberbullying among Middle School Students

Previous research has examined different factors relating to cyber bullying, but no one has specifically looked at how empathy, exploitative narcissism, and gender contribute to cyber bullying. We hypothesize that all three factors, empathy, exploitative narcissism, and gender, will uniquely contribute to cyber bullying. Middle schoolers (n = 851) completed surveys measuring each of these factors. A series of regression analyses found that exploitative narcissism and low empathy uniquely contribute to cyber bullying. However, there was not a significant finding for gender. The results from this analysis suggest that individuals who exhibit exploitative narcissism and low empathy are more likely to partake in cyber bullying.https://encompass.eku.edu/swps_undergraduategallery/1040/thumbnail.jp

Clinician Telehealth Attitudes in a Rural Community Mental Health Center Setting.

Telehealth-based services in community mental health settings are on the rise and growth is expected to continue. Negative clinician attitudes toward telehealth have been identified as a key barrier to overall telehealth acceptance and implementation. The present study examined rural clinical mental health staff members’ attitudes toward telehealth. One hundred clinicians participated in a mixed-methods, Internet-based survey. Eighty-nine percent of respondents reported a favorable or neutral opinion of telehealth and 100% of participants reported their agency provided one or more clinical services via telehealth. Clinicians identified telehealth-related concerns about their ability to establish therapeutic alliance, software and equipment usability, associated costs, whether telehealth-delivered services were equivalent to face-to-face treatment, and HIPAA. These concerns were in line with previous research and all represent areas where additional training or knowledge could potentially address clinician apprehension. We found a strong positive correlation, r .66, p .01 between telehealth knowledge and telehealth experience. Telehealth knowledge predicted telehealth opinion (β .430, R2 .19, p .01) and an agency’s technological capability to provide services via telehealth predicted clinicians’ willingness to consider providing services via telehealth (β .390, R2 .15, p .05). Researchers and trainers should focus on increasing knowledge about the effectiveness of telehealth and providing clinicians with safe opportunities to gain comfort and competency with the technology needed to provide these types of specialized services