A Four Marker Digital PCR Toolkit for Detecting Heavy Alcohol Consumption and the Effectiveness of Its Treatment.

Background.-Heavy alcohol consumption (HAC) is a shared concern of the forensic, medical and insurance underwriting communities. Unfortunately, there is a relative lack of clinically employable tools for detecting HAC and monitoring treatment response. Building on the results of 3 genome wide methylation studies, we have previously shown in a small group of samples that methylation sensitive digital PCR assays (MSdPCR) have the potential to accurately classify individuals with respect to HAC in a small set of individuals. Objective.-We now expand on those earlier findings using data and biomaterials from 143 participants with current HAC and 200 abstinent controls. Results.-We show that a set of 4 digital PCR assays that have a receiver operating characteristic (ROC) area under the curve (AUC) of 0.96 for detecting those with HAC. After a mean of 21 days of inpatient enforced abstinence, methylation status at one of these markers, cg04987734, began to revert to baseline values. Re-examination of methylation data from our smaller 2014 study with respect to this locus demonstrated a similarly significant reversion pattern at cg04987734 in association with treatment enforced abstinence. Conclusions.-We conclude that clinically implementable dPCR tools can sensitively detect the presence of HAC and that they show promise for monitoring alcohol treatment results. These dPCR tools could be useful to clinicians and researchers in monitoring those enrolled in substance use disorder treatment, employee wellness programs and insurance underwriting.

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.

The folding free-energy surface of HIV-1 protease: insights into the thermodynamic basis for resistance to inhibitors.

Spontaneous mutations at numerous sites distant from the active site of human immunodeficiency virus type 1 protease enable resistance to inhibitors while retaining enzymatic activity. As a benchmark for probing the effects of these mutations on the conformational adaptability of this dimeric beta-barrel protein, the folding free-energy surface of a pseudo-wild-type variant, HIV-PR(*), was determined by a combination of equilibrium and kinetic experiments on the urea-induced unfolding/refolding reactions. The equilibrium unfolding reaction was well described by a two-state model involving only the native dimeric form and the unfolded monomer. The global analysis of the kinetic folding mechanism reveals the presence of a fully folded monomeric intermediate that associates to form the native dimeric structure. Independent analysis of a stable monomeric version of the protease demonstrated that a small-amplitude fluorescence phase in refolding and unfolding, not included in the global analysis of the dimeric protein, reflects the presence of a transient intermediate in the monomer folding reaction. The partially folded and fully folded monomers are only marginally stable with respect to the unfolded state, and the dimerization reaction provides a modest driving force at micromolar concentrations of protein. The thermodynamic properties of this system are such that mutations can readily shift the equilibrium from the dimeric native state towards weakly folded states that have a lower affinity for inhibitors but that could be induced to bind to their target proteolytic sites. Presumably, subsequent secondary mutations increase the stability of the native dimeric state in these variants and, thereby, optimize the catalytic properties of the resistant human immunodeficiency virus type 1 protease.