Dose Response and Prediction Characteristics of a Methylation Sensitive Digital PCR Assay for Cigarette Consumption in Adults.

The tobacco use disorders are the largest preventable cause of morbidity and mortality in the world. A substantial barrier to the development of better intervention and screening measures is the lack of clinically employable biomarkers to detect the existence and extent of tobacco consumption. In prior work, we and others have shown that array based assessment of DNA methylation status at cg05575921 is a sensitive and quantitative method for assessing cigarette consumption. Unfortunately, in general, arrays are not practical clinical tools. Herein, we detail the prediction performance metrics and dose dependency of a clinically implementable droplet digital PCR (ddPCR) assay for cigarette consumption in adults. First, we demonstrate that measurements of cg05575921 as determined by Illumina array and ddPCR are highly correlated (R2 = 0.98, n = 92). Second, using clinical data and biomaterial from 177 subjects ranging from 18 to 78 years of age, we show that the Receiver Operating Characteristic (ROC) area under the curve (AUC) for classifying smoking status using methylation status at cg05575921 is 0.99. Finally, we conduct modeling analyses of cigarette consumption over discrete time periods to show that methylation status is best correlated with mean cigarette consumption over the past year (R2 = 0.5) and that demethylation at cg05575921 is dose dependent with a demethylation (delta beta) of 1% being equivalent to 1.2 cigarettes per day. But we do not find a relationship between Fagerstrom score and DNA methylation. We conclude that ddPCR assessment of cg05575921 methylation is an accurate method for assessing the presence and extent of cigarette consumption in adult subjects. We suggest that skillful clinical implementation of this approach alone or in combination with other assessment methods could lead to substantial reduction of cigarette consumption related morbidity and mortality.

Genome-wide and digital polymerase chain reaction epigenetic assessments of alcohol consumption.

The lack of readily employable biomarkers of alcohol consumption is a problem for clinicians and researchers. In 2014, we published a preliminary DNA methylation signature of heavy alcohol consumption that remits as a function of abstinence. Herein, we present new genome-wide methylation findings from a cohort of additional subjects and a meta-analysis of the data. Using DNA from 47 consecutive heavy drinkers admitted for alcohol detoxification in the context of alcohol treatment and 47 abstinent controls, we replicate the 2014 results and show that 21,221 CpG residues are differentially methylated in active heavy drinkers. Meta-analysis of all data from the 448,058 probes common to the two methylation platforms shows a similarly profound signature with confirmation of findings from other groups. Principal components analyses show that genome-wide methylation changes in response to alcohol consumption load on two major factors with one component accounting at least 50% of the total variance in both smokers and nonsmoking alcoholics. Using data from the arrays, we derive a panel of five methylation probes that classifies use status with a receiver operator characteristic area under the curve (AUC) of 0.97. Finally, using droplet digital polymerase chain reaction (PCR), we convert these array-based findings to two marker assays with an AUC of 0.95 and a four marker set AUC of 0.98. We conclude that DNA methylation assessments are capable of quantifying alcohol use status and suggest that readily employable digital PCR approaches for substance consumption may find widespread use in alcohol-related research and patient care.

A qualitative exploration of opinions on the community pharmacists' role amongst the general public in Scotland.

OBJECTIVE: To understand members of the public's opinions and experiences of pharmacy services. METHOD: This exploratory study employed qualitative methods. Five focus groups were conducted with 26 members of the public resident in Scotland in March 2010. The groups comprised those perceived to be users and non-users of community pharmacy. A topic guide was developed to prompt discussion. Each focus group was recorded, transcribed, anonymised and analysed using thematic analysis. KEY FINDINGS: Participants made positive comments about pharmacy services although many preferred to see a general practitioner (GP). Participants discussed using pharmacies for convenience, often because they were unable to access GPs. Pharmacists were perceived principally to be suppliers of medicine, although there was some recognition of roles in dealing with minor ailments and providing advice. For those with serious and long-standing health matters GPs were usually the professional of choice for most health needs. Community pharmacy was seen to offer incomplete services which did not co-ordinate well with other primary-care services. The pharmacy environment and retail setting were not considered to be ideal for private healthcare consultations. CONCLUSIONS: This study suggests that despite recent initiatives to extend the role of community pharmacists many members of the general public continue to prefer a GP-led service. Importantly GPs inspire public confidence as well as offering comprehensive services and private consultation facilities. Improved communication and information sharing between community pharmacists and general practice could support community pharmacist-role expansion.