Choice of gDNA isolation method has a significant impact on average murine Telomere Length estimates.

Telomere Length (TL) and integrity is significantly associated with age-related disease, multiple genetic and environmental factors. We observe mouse genomic DNA (gDNA) isolation methods to have a significant impact on average TL estimates. The canonical qPCR method does not measure TL directly but via the ratio of telomere repeats to a single copy gene (SCG) generating a T/S ratio. We use a monochromatic-multiplex-qPCR (mmqPCR) method which multiplexes the PCR and enables quantification of the target and the single copy gene within the same qPCR reaction. We demonstrate that TL measurements, from murine gDNA, isolated via Spin Columns (SC) and Magnetic Beads (MB), generate significantly smaller T/S ratios compared to gDNA isolated via traditional phenol/chloroform methods. The former methods may impede correct TL estimation by producing non representative fragment sets and reducing qPCR efficacy. This work highlights discrepancies in TL measurements due to different extraction techniques. We recommend the use of gDNA isolation methods that are shown to preserve DNA length and integrity, such as phenol/chloroform isolation. We propose that widely used high throughput DNA isolation methodologies can create spurious associations within a sample set, thus creating misleading data. We suggest that published TL associations should be revisited in the light of these data.

Cannabis use frequency and pain interference among people with HIV.

Cannabis is often used by people with HIV (PWH) for pain, yet study results are inconsistent regarding whether and how it affects pain. This study examines whether greater cannabis use frequency is associated with lower pain interference and whether cannabis use modifies the association of pain severity and pain interference among 134 PWH with substance dependence or a lifetime history of injection drug use. Multi-variable linear regression models examined the association between past 30-day cannabis use frequency and pain interference. Additional models evaluated whether cannabis use modified the association between pain severity and pain interference. Cannabis use frequency was not significantly associated with pain interference. However, in a model with interaction between cannabis use frequency and pain severity, greater cannabis use frequency attenuated the strength of the association between pain severity and pain interference (p = 0.049). The adjusted mean difference (AMD) in pain interference was +1.13, + 0.81, and +0.05 points for each 1-point increase in pain severity for those with no cannabis use, 15 days of use, and daily use, respectively. These findings suggest that attenuating the impact of pain severity on pain-related functional impairment is a potential mechanism for a beneficial role of cannabis for PWH.

Medication for addiction treatment and acute care utilization in HIV-positive adults with substance use disorders.

Medication for addiction treatment (MAT) could reduce acute care utilization in HIV-positive individuals with substance use disorders. The study objective was to determine if HIV-positive people with substance use disorders treated with MAT report less acute care utilization than those not receiving MAT. We assessed the association between MAT and acute care utilization among HIV-positive individuals with alcohol or opioid use disorder. Acute care utilization 6 months later was defined as any past 3-month self-reported (1) emergency department (ED) visit and (2) hospitalization. Of 153 participants, 88% had alcohol use disorder, 41% had opioid use disorder, and 48 (31%) were treated with MAT. Fifty-five (36%) participants had an ED visit and 38 (25%) participants had a hospitalization. MAT was not associated with an ED visit (AOR 1.12, 95% CI 0.46-2.75) or hospitalization (AOR 1.09, 95% CI 0.39-3.04). MAT was not associated with acute care utilization. These results highlight the need to increase MAT prescribing in HIV-positive individuals with substance use disorders, and to address the many factors that influence acute care utilization.

Evolution of illicit opioid use among people with HIV infection in St Petersburg, Russia, in the period 2004-2015.

OBJECTIVES: In the late 1990s, when the current Russian opioid epidemic began, illicit opioids used in Russia consisted almost exclusively of heroin. The type of opioids used has evolved in the early 21st Century. The objective of this study was to describe the evolution of illicit opioid use among people living with HIV (PLWH) reporting recent opioid use in St Petersburg, Russia. METHODS: We examined baseline data from four research studies conducted in the period 2004-2015 that included PLWH who used opioids [Partnership to Reduce the Epidemic Via Engagement in Narcology Treatment (PREVENT; 2004-2005; n = 17), HIV Evolution in Russia-Mitigating Infection Transmission and Alcoholism in a Growing Epidemic (HERMITAGE; 2007-2010; n = 281), Linking Infectious and Narcology Care (LINC; 2013-2014; n = 119) and Russia Alcohol Research Collaboration on HIV/AIDS (Russia ARCH; 2012-2015; n = 121)] and reported recent use of heroin and other opioids. RESULTS: Although these studies spanned more than a decade, the participants represented similar birth cohorts; the mean age was 24.5 years in 2004 and 33.3 years in 2014. The use of opioid types, however, evolved across cohorts, with the use of any illicit drug other than heroin increasing from 6% [95% confidence interval (CI) 000.2, 29%] in PREVENT (2004-2005) to 30% (95% CI 25, 36%) in HERMITAGE (2007-2010) to 70% (95% CI 61, 78%) in LINC (2013-2014) to 77% (95% CI 68, 84%) in ARCH (2012-2015). Any heroin use consistently decreased over the 10-year period in the cohorts, from 100% (95% CI 80, 100%) in 2004-2005 to 54% (95% CI 44, 63%) in 2012-2015. CONCLUSIONS: Among PLWH who use opioids in St Petersburg, Russia, illicit use of opioids other than heroin appears to be more common than heroin use.

Macrophage gene expression in adipose tissue is associated with insulin sensitivity and serum lipid levels independent of obesity.

OBJECTIVE: Obesity is linked to both increased metabolic disturbances and increased adipose tissue macrophage infiltration. However, whether macrophage infiltration directly influences human metabolism is unclear. The aim of this study was to investigate if there are obesity-independent links between adipose tissue macrophages and metabolic disturbances. DESIGN AND METHODS: Expression of macrophage markers in adipose tissue was analyzed by DNA microarrays in the SOS Sib Pair study and in patients with type 2 diabetes and a BMI-matched healthy control group. RESULTS: The expression of macrophage markers in adipose tissue was increased in obesity and associated with several metabolic and anthropometric measurements. After adjustment for BMI, the expression remained associated with insulin sensitivity, serum levels of insulin, C-peptide, high density lipoprotein cholesterol (HDL-cholesterol) and triglycerides. In addition, the expression of most macrophage markers was significantly increased in patients with type 2 diabetes compared to the control group. CONCLUSION: Our study shows that infiltration of macrophages in human adipose tissue, estimated by the expression of macrophage markers, is increased in subjects with obesity and diabetes and associated with insulin sensitivity and serum lipid levels independent of BMI. This indicates that adipose tissue macrophages may contribute to the development of insulin resistance and dyslipidemia.

Sexually transmitted infections among HIV-infected heavy drinkers in St Petersburg, Russia.

The objective of this study was to estimate the prevalence and identify correlates of four sexually transmitted infections (STIs) among HIV-infected Russians reporting heavy alcohol use and recent unprotected sex, we conducted a cross-sectional analysis of baseline data from the HERMITAGE study. The primary outcome was any current STI, based on urine tests for Neisseria gonorrhoeae, Chlamydia trachomatis and Trichomonas vaginalis and serological testing for infection with Treponema pallidum. Data on potential demographic and behavioural predictors of STI were obtained from surveys administered at study entry. Of 682 participants, 12.8% (95% confidence interval [CI] 10.3, 15.3) tested positive for at least one STI. In a multivariable model adjusted for gender, age and marital status, only sex trade involvement over the last three months was significantly associated with an increased odds of STI (adjusted odds ratio [AOR] 2.00, 95% CI 1.13, 3.55). Given that STIs were common in this HIV-infected cohort, and that few patient characteristics predicted STI, the current practice of screening HIV-infected Russians for syphilis alone merits re-evaluation.

Candidate DNA methylation drivers of acquired cisplatin resistance in ovarian cancer identified by methylome and expression profiling.

Multiple DNA methylation changes in the cancer methylome are associated with the acquisition of drug resistance; however it remains uncertain how many represent critical DNA methylation drivers of chemoresistance. Using isogenic, cisplatin-sensitive/resistant ovarian cancer cell lines and inducing resensitizaton with demethylating agents, we aimed to identify consistent methylation and expression changes associated with chemoresistance. Using genome-wide DNA methylation profiling across 27 578 CpG sites, we identified loci at 4092 genes becoming hypermethylated in chemoresistant A2780/cp70 compared with the parental-sensitive A2780 cell line. Hypermethylation at gene promoter regions is often associated with transcriptional silencing; however, expression of only 245 of these hypermethylated genes becomes downregulated in A2780/cp70 as measured by microarray expression profiling. Treatment of A2780/cp70 with the demethylating agent 2-deoxy-5'-azacytidine induces resensitization to cisplatin and re-expression of 41 of the downregulated genes. A total of 13/41 genes were consistently hypermethylated in further independent cisplatin-resistant A2780 cell derivatives. CpG sites at 9 of the 13 genes (ARHGDIB, ARMCX2, COL1A, FLNA, FLNC, MEST, MLH1, NTS and PSMB9) acquired methylation in ovarian tumours at relapse following chemotherapy or chemoresistant cell lines derived at the time of patient relapse. Furthermore, 5/13 genes (ARMCX2, COL1A1, MDK, MEST and MLH1) acquired methylation in drug-resistant ovarian cancer-sustaining (side population) cells. MLH1 has a direct role in conferring cisplatin sensitivity when reintroduced into cells in vitro. This combined genomics approach has identified further potential key drivers of chemoresistance whose expression is silenced by DNA methylation that should be further evaluated as clinical biomarkers of drug resistance.

Differential coexpression analysis of obesity-associated networks in human subcutaneous adipose tissue.

OBJECTIVE: To use a unique obesity-discordant sib-pair study design to combine differential expression analysis, expression quantitative trait loci (eQTLs) mapping and a coexpression regulatory network approach in subcutaneous human adipose tissue to identify genes relevant to the obese state. STUDY DESIGN: Genome-wide transcript expression in subcutaneous human adipose tissue was measured using Affymetrix U133 Plus 2.0 microarrays (Affymetrix, Santa Clara, CA, USA), and genome-wide genotyping data was obtained using an Applied Biosystems (Applied Biosystems; Life Technologies, Carlsbad, CA, USA) SNPlex linkage panel. SUBJECTS: A total of 154 Swedish families ascertained through an obese proband (body mass index (BMI) >30 kg m(-2)) with a discordant sibling (BMI>10 kg m(-2) less than proband). RESULTS: Approximately one-third of the transcripts were differentially expressed between lean and obese siblings. The cellular adhesion molecules (CAMs) KEGG grouping contained the largest number of differentially expressed genes under cis-acting genetic control. By using a novel approach to contrast CAMs coexpression networks between lean and obese siblings, a subset of differentially regulated genes was identified, with the previously GWAS obesity-associated neuronal growth regulator 1 (NEGR1) as a central hub. Independent analysis using mouse data demonstrated that this finding of NEGR1 is conserved across species. CONCLUSION: Our data suggest that in addition to its reported role in the brain, NEGR1 is also expressed in subcutaneous adipose tissue and acts as a central 'hub' in an obesity-related transcript network.

Positive replication and linkage disequilibrium mapping of the chromosome 21q22.1 malaria susceptibility locus.

Four cytokine receptor genes are located on Chr21q22.11, encoding the alpha and beta subunits of the interferon-alpha receptor (IFNAR1 and IFNAR2), the beta subunit of the interleukin 10 receptor (IL10RB) and the second subunit of the interferon-gamma receptor (IFNGR2). We previously reported that two variants in IFNAR1 were associated with susceptibility to malaria in Gambians. We now present an extensive fine-scale mapping of the associated region utilizing 45 additional genetic markers obtained from public databases and by sequencing a 44 kb region in and around the IFNAR1 gene in 24 Gambian children (12 cases/12 controls). Within the IFNAR1 gene, a newly studied C --> G single-nucleotide polymorphism (IFNAR1 272354c-g) at position -576 relative to the transcription start was found to be more strongly associated with susceptibility to severe malaria. Association was observed in three populations: in Gambian (P=0.002), Kenyan (P=0.022) and Vietnamese (P=0.005) case-control studies. When all three studies were combined, using the Mantel-Haenszel test, the presence of IFNAR1 -576G was associated with a substantially elevated risk of severe malaria (N=2444, OR=1.38, 95% CI: 1.17-1.64; P=1.7 x 10(-4)). This study builds on previous work to further highlight the importance of the type-I interferon pathway in malaria susceptibility and illustrates the utility of typing SNPs within regions of high linkage disequilibrium in multiple populations to confirm initial positive associations.

Illicit drugs, alcohol, and addiction in human immunodeficiency virus.

Drug and alcohol use complicate both the prevention and treatment of human immunodeficiency virus (HIV) infection. Substance use is one of the major engines driving HIV transmission, directly, through the sharing of injection drug use equipment and indirectly, through increasing risky sexual behaviors. Drug and alcohol dependence compromise effective HIV treatment by influencing both access and adherence to antiretroviral therapy. Exposure to addictive substances may have direct immunosuppressive effects independent of their impact on access and adherence to treatment. Measures effective at minimizing HIV transmission attributable to drug and alcohol use include HIV testing and referral to treatment, syringe and needle exchange programs, opioid replacement therapy (i.e., methadone and buprenorphine), and behavioral interventions targeting HIV risk behaviors among both HIV-infected and HIV-uninfected people. Measures effective at optimizing HIV treatment among alcohol and drug-dependent patients include HIV testing with referral to treatment and substance use treatment that is linked to or integrated into HIV treatment. Due to the intertwining problems of substance use and HIV infection, physicians and other health care providers must address the issues of illicit drugs and alcohol use as mainstream medical problems in order to provide optimal care for HIV-infected patients.

Single nucleotide polymorphisms in the neuropeptide Y2 receptor (NPY2R) gene and association with severe obesity in French white subjects.

AIMS/HYPOTHESIS: Genetic variants of genes for peptide YY (PYY), neuropeptide Y2 receptor (NPY2R) and pancreatic polypeptide (PPY) were investigated for association with severe obesity. SUBJECTS AND METHODS: The initial screening of the genes for variants was performed by sequencing in a group of severely obese subjects (n=161). Case-control analysis of the common variants was then carried out in 557 severely obese adults, 515 severely obese children and 1,163 non-obese/non-diabetic control subjects. Rare variants were genotyped in 700 obese children and the non-obese/non-diabetic control subjects (n=1,163). RESULTS: Significant association was found for a 5' variant (rs6857715) in the NPY2R gene with both severe adult obesity (p=0.002) and childhood obesity (p=0.02). This significant association was further supported by a pooled allelic analysis of all obese cases (adults and children, n=928) vs the control subjects (n=938) (p=0.0004, odds ratio=1.3, 95% CI 1.1-1.5). Quantitative trait analysis of BMI and WHR was performed and significant association was observed for SNP rs1047214 in NPY2R with an increase in WHR in the severely obese children (co-dominant model p=0.005, recessive model p=0.001). Association was also observed for an intron 3 variant (rs162430) in the PYY gene with childhood obesity (p=0.04). No significant associations were observed for PPY variants. Only one rare variant in the NPY2R gene (C-5641T) was not found in lean individuals and this was found to co-segregate with obesity in one family. CONCLUSIONS/INTERPRETATION: These results provide evidence of association for NPY2R and PYY gene variants with obesity and none for PPY variants. A rare variant of the NPY2R gene showed evidence of co-segregation with obesity and its contribution to obesity should be investigated further.

Interferon-alpha receptor-1 (IFNAR1) variants are associated with protection against cerebral malaria in the Gambia.

The chromosome 21q22.11 cytokine receptor cluster contains four genes that encode subunits of the receptors for the cytokines interleukin-10 and interferon-alpha, -beta and -gamma that may have a role in malaria pathogenesis. A total of 15 polymorphic markers located within these genes were initially genotyped in 190 controls and 190 severe malaria cases from The Gambia. Two interferon-alpha receptor-1 (IFNAR1) gene SNPs (17470 and L168 V) showed evidence for an association with severe malaria phenotypes and were typed in a larger series of samples comprising 538 severe malaria cases, 338 mild malaria cases and 562 controls. Both the 17470-G/G and L168V-G/G genotypes were associated with protection against severe malaria, in general, and cerebral malaria, in particular (P=0.004 and 0.003, respectively). IFNAR1 diplotypes were then constructed for these two markers using the PHASE software package. The (17470-G L168V-G/17470-G L168V-G) diplotype was found to be associated with a reduced risk of cerebral malaria and the (17470-C L168V-C/17470-G L168V-G) diplotype with an increased risk of cerebral malaria (overall 3 x 2 chi(2)=12.8, d.f.=2, P=0.002 and 3 x 2 chi(2)=15.2, d.f.=2, P=0.0005, respectively). These data suggest a role for the type I interferon pathway in resistance to cerebral malaria.

Gene polymorphism in Netherton and common atopic disease.

Atopic dermatitis (AD) and asthma are characterized by IgE-mediated atopic (allergic) responses to common proteins (allergens), many of which are proteinases. Loci influencing atopy have been localized to a number of chromosomal regions, including the chromosome 5q31 cytokine cluster. Netherton disease is a rare recessive skin disorder in which atopy is a universal accompaniment. The gene underlying Netherton disease (SPINK5) encodes a 15-domain serine proteinase inhibitor (LEKTI) which is expressed in epithelial and mucosal surfaces and in the thymus. We have identified six coding polymorphisms in SPINK5 (Table 1) and found that a Glu420-->Lys variant shows significant association with atopy and AD in two independent panels of families. Our results implicate a previously unrecognized pathway for the development of common allergic illnesses.

Linkage and allelic association of chromosome 5 cytokine cluster genetic markers with atopy and asthma associated traits.

Linkage and association of polymorphic markers in the chromosome 5q31-q33 cytokine cluster to atopy and asthma associated phenotypes have been reported by a number of groups. To investigate this region, 29 polymorphic markers were used to genotype a combined set of 233 families. These markers were ordered based upon the genetic data, supplemented by published genetic and physical maps. Significant two-point linkage was observed for asthma (most significant marker IRF1, P = 0.0002) and atopy (CD14SNP, P = 0.0001). Allelic association was observed between D5S463 and atopy (P = 0.002) and the skin prick test index (P = 0.04). The data support the possibility of three asthma/atopy loci in the 5q31-q33 region, each with a relatively small effect.

Genetic linkage of childhood atopic dermatitis to psoriasis susceptibility loci.

We have carried out a genome screen for atopic dermatitis (AD) and have identified linkage to AD on chromosomes 1q21, 17q25 and 20p. These regions correspond closely with known psoriasis loci, as does a previously identified AD locus on chromosome 3q21. The results indicate that AD is influenced by genes with general effects on dermal inflammation and immunity.

Delta 32 deletion of CCR5 gene and association with asthma or atopy.

The CCR5-delta32 deletion polymorphism (CCR5-delta32) was investigated for linkage and association to asthma and atopy using two panels of nuclear families containing 1284 individuals. No statistically significant linkage to asthma/wheeze or atopy was observed in either of the two panels of families. Multiallelic transmission disequilibrium tests (TDT) of the combined data found no significant association for atopy (52 independent alleles transmitted, 51 non-transmitted) or asthma/wheeze (39 transmitted, 44 non-transmitted). Although functional evidence might suggest that CCR5 is a good candidate gene for atopic asthma, this study provides no genetic evidence from CCR5-delta32 polymorphism to support this hypothesis.

Older, hypertensive, and hypercholesterolemic fairgoers visit more booths and differ in their health concerns at a community health fair.

The purpose of this study was to determine whether persons attending a community health fair had different health concerns and booth visitation patterns based on their risk factor profiles. All fairgoers were encouraged to complete an anonymous survey of demographic information, top 4 health concerns, and selected cardiac risk factors. Over the five-hour duration of the fair, 329 surveys were collected from about 450 fairgoers. There were no exclusion criteria for the survey. The fair was sponsored by the Maryland Chapter of the American College of Physicians, organized by medical students from the University of Maryland and Johns Hopkins University, and included 23 booths on a variety of health topics. Older fairgoers and fairgoers with a self-reported history of high blood pressure or elevated cholesterol showed an increased interest in hypertension and heart disease (p < 0.05). Older fairgoers also showed an increased interest in health topics related to aging, such as estrogen replacement therapy and geriatric medicine. Older, hypertensive and hypercholesterolemic fairgoers visited an increased mean number of total booths when compared to other respondents (p < 0.05). Most booths reported a higher percentage of older, hypertensive, and hypercholesterolemic visitors than the overall percentage of fairgoers who reported these risk factors. These results suggest that booth visitation patterns of health fair participants may be viewed as a deliberate attempt by at risk populations to access health information particular to their needs.