A series of subclonal cell lines with high or low differentiation/mineralization potential after growth in the presence of ascorbic acid (AA) were derived from murine MC3T3-E1 cells. Subclones were characterized in terms of their ability to mineralize a collagenous extracellular matrix both in vitro and in vivo and express osteoblast-related genes. When compared with nonmineralizing cells, mineralizing subclones selectively expressed mRNAs for the osteoblast markers, bone sialoprotein (BSP), osteocalcin (OCN), and the parathyroid hormone (PTH)/parathyroid hormone-related protein (PTHrP) receptor. In contrast, alkaline phosphatase mRNA was present in certain nonmineralizing as well as mineralizing subclones, suggesting that its expression may be subject to different controls from other osteoblast markers. Only highly differentiating subclones exhibited strong AA-dependent induction of a transiently transfected OCN promoter-luciferase reporter gene, indicating that there was a good correlation between mRNA levels and transcriptional activity. Consistent with its postulated role in biomineralization, BSP as measured by Western blotting was only present in mineralizing subclones. After implantation into immunodeficient mice, highly differentiating subclones formed bone-like ossicles resembling woven bone, while poorly differentiating cells only produced fibrous tissue. Interestingly, subclones with both high and low differentiation potential produced similar amounts of collagen in culture and expressed comparable basal levels of mRNA encoding Osf2/Cbfa1, an osteoblast-related transcription factor. Although some strongly differentiating cells exhibited a modest AA-dependent up-regulation of Osf2/Cbfa1 mRNA, there was no clear relationship between levels of this message and induction of mRNAs for other differentiation markers. Thus, the mere presence of Osf2/Cbfa1 in a subclone was not sufficient for osteoblast differentiation. These subclones will be very useful for studying critical events in osteoblast differentiation and mineralization. (J Bone Miner Res 1999;14:893-903)

Background Whole-genome sequencing (WGS) in asymptomatic adults might prevent disease but increase healthcare utilization without clinical value. Objective Describe the effect on clinical care and outcomes of adding WGS to standardized family history assessment in primary care. Design Pilot randomized trial. Setting Academic primary care practices. Participants Nine primary care physicians (PCPs) and 100 generally healthy patients aged 40–65. Interventions Patients were randomly assigned to receive a family history report alone (FH arm) or in combination with an interpreted WGS report including monogenic disease risk (MDR) results (associated with Mendelian disorders), carrier variants, pharmacogenomic associations, and polygenic risk estimates for cardiometabolic traits (FH+WGS arm). Each patient met with his or her PCP to discuss the reports. Measurements Clinical outcomes and healthcare utilization through six months were obtained from audio-recorded PCP-patient discussions and medical records. Patients’ health behavior changes were surveyed six months after receiving results. A panel of clinician-geneticists rated the appropriateness of how PCPs managed MDR results. Results Mean age was 55 years; 58% were female. Eleven FH+WGS patients (22%, 12%–36%) had new MDR results. Only two (4%, 0.01%–14%) had evidence of the phenotypes predicted by an MDR result (fundus albipunctatus due to RDH5 and variegate porphyria due to PPOX). PCPs recommended new clinical actions for 16% (8%–30%) of FH patients and 34% (22%–49%) of FH+WGS patients. Thirty (17%–45%) and 41% (27%–56%) of FH and FH+WGS patients, respectively, reported making a health behavior change after six months. Geneticists rated PCP management of eight MDR results (73%, 39%–99%) as appropriate and two (18%, 3%–52%) as inappropriate. Limitations Limited sample size and ancestral and socioeconomic diversity. Conclusions Adding WGS to primary care reveals new molecular findings of uncertain clinical utility. Non-geneticist providers may be able to manage WGS results appropriately, but WGS may prompt additional clinical actions of unclear value. Registration ClinicalTrials.gov identifier NCT01736566 Funding National Institutes of Health

Communicating genetic risk information in ways that maximize understanding and promote health is increasingly important given the rapidly expanding availability and capabilities of genomic technologies. A well-developed literature on risk communication in general provides guidance for best practices, including presentation of information in multiple formats, attention to framing effects, use of graphics, sensitivity to the way numbers are presented, parsimony of information, attentiveness to emotions, and interactivity as part of the communication process. Challenges to communicating genetic risk information include deciding how best to tailor it, streamlining the process, deciding what information to disclose, accepting that communications may have limited influence, and understanding the impact of context. Meeting these challenges has great potential for empowering individuals to adopt healthier lifestyles and improve public health, but will require multidisciplinary approaches and collaboration.

BackgroundWhole genome sequencing (WGS) is already being used in certain clinical and research settings, but its impact on patient well-being, health-care utilization, and clinical decision-making remains largely unstudied. It is also unknown how best to communicate sequencing results to physicians and patients to improve health. We describe the design of the MedSeq Project: the first randomized trials of WGS in clinical care.Methods/DesignThis pair of randomized controlled trials compares WGS to standard of care in two clinical contexts: (a) disease-specific genomic medicine in a cardiomyopathy clinic and (b) general genomic medicine in primary care. We are recruiting 8 to 12 cardiologists, 8 to 12 primary care physicians, and approximately 200 of their patients. Patient participants in both the cardiology and primary care trials are randomly assigned to receive a family history assessment with or without WGS. Our laboratory delivers a genome report to physician participants that balances the needs to enhance understandability of genomic information and to convey its complexity. We provide an educational curriculum for physician participants and offer them a hotline to genetics professionals for guidance in interpreting and managing their patients’ genome reports. Using varied data sources, including surveys, semi-structured interviews, and review of clinical data, we measure the attitudes, behaviors and outcomes of physician and patient participants at multiple time points before and after the disclosure of these results.DiscussionThe impact of emerging sequencing technologies on patient care is unclear. We have designed a process of interpreting WGS results and delivering them to physicians in a way that anticipates how we envision genomic medicine will evolve in the near future. That is, our WGS report provides clinically relevant information while communicating the complexity and uncertainty of WGS results to physicians and, through physicians, to their patients. This project will not only illuminate the impact of integrating genomic medicine into the clinical care of patients but also inform the design of future studies.Trial registrationClinicalTrials.gov identifier NCT01736566

Background Although the integration of whole genome sequencing (WGS) into standard medical practice is rapidly becoming feasible, physicians may be unprepared to use it. Methods Primary care physicians (PCPs) and cardiologists enrolled in a randomized clinical trial of WGS received genomics education before completing semi-structured interviews. Themes about preparedness were identified in transcripts through team-based consensus-coding. Results Data from eleven PCPs and nine cardiologists suggested that physicians enrolled in the trial primarily to prepare themselves for widespread use of WGS in the future. PCPs were concerned about their general genomic knowledge, while cardiologists were concerned about how to interpret specific types of results and secondary findings. Both cohorts anticipated preparing extensively before disclosing results to patients by using educational resources with which they were already familiar, and both cohorts anticipated making referrals to genetics specialists as needed. A lack of laboratory guidance, time pressures, and a lack of standards contributed to feeling unprepared. Discussion Physicians had specialty-specific concerns about their preparedness to use WGS. Findings identify specific policy changes that could help physicians feel more prepared, and highlight how providers of all types will need to become familiar with interpreting WGS results. Clinical Trials Registration ClinicalTrials.gov # NCT01736566.

Bronchoalveolar lavage has been widely used to sample the lower respiratory tract. Most of the material recovered with this technique represents alveolar contents. A number of modifications have been suggested in order to obtain samples relatively enriched for bronchial material. In order to be able to use a standard technique for bronchoalveolar lavage to sample both airways and "routine" alveolar material, a simple modification of the technique as described by Reynolds and Newball was used: five sequential 20-ml aliquots were infused into the lower respiratory tract, and each aliquot was immediately aspirated. The return from the first aliquot was processed separately from the return from the subsequent four aliquots. These last four aliquots were pooled. Analysis of the first aliquot revealed it to be enriched for ciliated epithelial cells when compared with the subsequent aliquots. There were also differences in inflammatory cell composition with the bronchial sample containing relatively more neutrophils and relatively less lymphocytes. Aspiration during transoral bronchoscopy was documented by quantifying salivary amylase in the bronchial and alveolar lavage fluids. It was estimated, however, that the aspiration was not of quantitative significance in the vast majority of subjects studied. Finally, with the technique of fractional processing of bronchoalveolar lavage samples, it was possible to compare the protein concentrations in bronchial and alveolar lavages. Most prominent among the differences was a marked relative enrichment in the bronchial samples for immunoglobulin A. The technique of fractional processing of bronchoalveolar lavage samples provides a simple means to obtain samples enriched for bronchial and alveolar components. This should facilitate analysis of lower respiratory tract specimens in airway disease.

Purpose Direct-to-consumer genetic testing is a new means of obtaining genetic testing outside of a traditional clinical setting. This study assesses genetic counselors’ experience, knowledge, and beliefs regarding direct-to-consumer genetic testing for tests that would currently be offered in genetics clinics. Methods Members of the National Society of Genetic Counselors completed a web-administered survey in February 2008. Results Response rate was 36%; the final data analysis included 312 respondents. Eighty-three percent of respondents had two or fewer inquiries about direct-to-consumer genetic testing, and 14% had received requests for test interpretation or discussion. Respondents believed that genetic counselors have a professional obligation to be knowledgeable about direct-to-consumer genetic testing (55%) and interpret results (48%). Fifty-one percent of respondents thought genetic testing should be limited to a clinical setting; 56% agreed direct-to-consumer genetic testing is acceptable if genetic counseling is provided. More than 70% of respondents would definitely or possibly consider direct-to-consumer testing for patients who (1) have concerns about genetic discrimination, (2) want anonymous testing, or (3) have geographic constraints. Conclusions Results indicate that genetic counselors have limited patient experiences with direct-to-consumer genetic testing and are cautiously considering if and under what circumstances this approach should be used

BackgroundThe MedSeq Project is a randomized clinical trial developing approaches to assess the impact of integrating genome sequencing into clinical medicine. To facilitate the return of results of potential medical relevance to physicians and patients participating in the MedSeq Project, we sought to develop a reporting approach for the effective communication of such findings.MethodsGenome sequencing was performed on the Illumina HiSeq platform. Variants were filtered, interpreted, and validated according to methods developed by the Laboratory for Molecular Medicine and consistent with current professional guidelines. The GeneInsight software suite, which is integrated with the Partners HealthCare electronic health record, was used for variant curation, report drafting, and delivery.ResultsWe developed a concise 5–6 page Genome Report (GR) featuring a single-page summary of results of potential medical relevance with additional pages containing structured variant, gene, and disease information along with supporting evidence for reported variants and brief descriptions of associated diseases and clinical implications. The GR is formatted to provide a succinct summary of genomic findings, enabling physicians to take appropriate steps for disease diagnosis, prevention, and management in their patients.ConclusionsOur experience highlights important considerations for the reporting of results of potential medical relevance and provides a framework for interpretation and reporting practices in clinical genome sequencing.Electronic supplementary materialThe online version of this article (doi:10.1186/s12881-014-0134-1) contains supplementary material, which is available to authorized users.