Peripheral Blood Cell DNA Research Articles

8-Hydroxy-2'-Deoxyguanosin in Peripheral Leukocyte Associated With HBsAg in Patients with Chronic Hepatitis B

Background: Chronic hepatitis B (CHB), with accumulation of Hepatitis B surface Antigen (HBsAg) in hepatocytes, linked to the immune-mediated hepatic inflammation and induction of oxidative stress. 8-Hydroxyl-2'-deoxyguanosine (8-OHdG) is a useful biomarker for measuring the adverse effects of exogenous infectious agents in oxidative damage to DNA. Objectives: The current study aimed at investigating the possible oxidative adverse effects of HBsAg and systemic DNA damage in patients with CHB, and supporting the host-viral interaction in immune-mediated inflammatory. Methods: Thirty patients with CHB who had undergone liver biopsies for therapeutic purposes and 30 matched controls from a healthy population were randomly selected in the present study for assessment of 8-OHdG levels in peripheral blood leukocytes DNA by 32P-postlabeling analysis. Expression of HBsAg in hepatocytes was evaluated immunohistochemically in liver biopsies of patients with CHB. The effect of 8-OHdG and 95% confidence interval (CI), adjusted by relevant confounders, were assessed on hepatitis B virus (HBV) infection. Results: Experimental investigation showed increased levels of DNA adduct 8-OHdG compared with healthy individuals (mean ± standard deviation, 1456 ± 1275 vs. 402 ± 271; P < 0.001). The logistic regression with continuous and dichotomous models revealed the strong impact of 8-OHdG on CHB infection (OR = 1.20; 95%CI: 1.01 - 1.44, P = 0.043) and (OR = 7.18; 95%CI: 1.32 - 39.02, P = 0.022). HBV DNA and hepatic expression of HBsAg had a borderline association with DNA adduct 8-OHdG (r = 0.35, P = 0.054 and r = 0.36, P = 0.05). Conclusions: The current study showed that the adduct of 8-OHdG in peripheral blood cells DNA increased in patients with CHB compared with healthy carriers and the pathophysiologic role of HBsAg in oxidative stress in patients with CHB. Nonetheless, the lack of efficient DNA repair enzymes activity as well as a proper diet with antioxidant agents in CHB need to be clarified in future studies.

Correlation between HFE gene polymorphisms and increased risk of coronary artery disease among patients with type 2 diabetes in Iran.

Diabetes mellitus is a risk factor for cardiovascular diseases (CVDs), which are among the major causes of deaths in type 2 diabetes (T2D). The purpose of the present study was to determine the association of C282Y and H63D mutations in the HFE gene with increased risk of coronary artery disease (CAD) in T2D patients. Two hundred and ninety individuals were divided into two groups: a case group and a control group. Genomic DNA of peripheral venous blood cells was extracted and the HFE gene mutations were analyzed using the PCR-RFLP technique. Data analysis revealed a significant difference between the allele frequencies of H63D and C282Y mutations between the case group and the controls (P < 0.05). The relationships between the GA and GG genotypes in C282Y and H63D mutations in terms of fasting blood sugar (FBS), lipid profile (total cholesterol, triglycerides, high-density lipoproteins (HDL), low-density lipoproteins), body mass index (BMI), HbA1c, micro albuminuria, and creatine levels did not show a significant differences between the two groups (P > 0.05). Using a logistic regression model, BMI, FBS, HDL, and total cholesterol levels were significantly different with independent predictors of CVD (P < 0.05). Our results revealed a significant correlation between C282Y and H63D mutations and the development of CAD in T2D patients.

Protective effects of spray-dried açaí (Euterpe oleracea Mart) fruit pulp against initiation step of colon carcinogenesis

The present study aims to evaluate whether açaí (Euterpe oleracea Mart) fruit pulp powder (AP) feeding attenuates the initiation step of chemically-induced mouse colon carcinogenesis. Male Swiss mice were fed low fat diet containing 2.5% or 5.0% of AP (weeks 1 to 4) produced by a spray-drying system. All groups received an intraperitoneal (i.p.) injection of colon carcinogen azoxymethane (AOM, 15mg/kg of body weight) at week 3. After the first administration of carcinogen, blood samples were collected to perform Single Cell Gel Electrophoresis Assay. Some mice were euthanized at week 3 (n=5 mice/group) and liver samples were collected for immunohistochemical and glutathione analysis. Other mice received a second i.p. injection of AOM at week 4 and were fed a high-fat diet to accelerate the development of preneoplastic aberrant crypt foci (ACF) until week 14 (n=10 mice/group). Cyanidin 3-glucoside and cyanidin 3-rutinoside, lutein, α-carotene and β-carotene were identified as the main anthocyanins and carotenoids in AP, respectively. At week 3, both dietary AP interventions (2.5% or 5.0%) reduced (p<0.001) peripheral blood cell DNA damage induced by AOM. Moreover, dietary 5.0% AP increased (p=0.002) hepatic total glutathione. At week 14, 5.0% AP intake reduced (p<0.05) ACF multiplicity. The findings indicate that AP feeding attenuates chemically-induced mouse colon carcinogenesis by increasing total GSH and attenuating DNA damage and preneoplastic lesion development.

Abstract 1068: Correlation of DNA methylation pattern between peripheral blood cell and normal breast tissue

Abstract Aberrant DNA methylation in peripheral blood cells (PBCs) may be a potential noninvasive biomarker for breast cancer risk. Whether methylations at CpG sites in PBCs can serve as proxies in normal breast tissues are unknown. We evaluated the correlation of DNA methylation between PBC DNA and breast cancer adjacent normal tissue using data and samples from the Shanghai Breast Cancer Study. Genome-wide DNA methylation profiles in PBCs and matched tumor adjacent normal tissues from 31 breast cancer patients were assayed using Illumina HumanMethylation450 BeadChip, which includes &amp;gt;485,000 CpG sites and covers 99% of RefSeq genes with multiple probes per gene. After quality control filtering, a total of 432,513 CpG sites were retained for analysis. We evaluated the association of methylation for each CpG site between DNA samples for PBCs and normal breast tissue using Spearman's rank correlation (r). The overall median correlation (r) is 0.35. A total of 99,928 (23.1%) and 18,290 (4.2%) of CpG sites had a high correlation between PBC DNA and normal breast tissue DNA with r &amp;gt; 0.50 and r &amp;gt; 0.75, respectively. Interestingly, we found that the correlation was negatively associated with both methylation levels (mean) and inter-person variability (standard deviation) in normal breast tissues (ρ = -0.14 and -0.19; P &amp;lt; 2.2 x10-16 for both). In 926 previously reported methylation quantitative trait loci, the correlations between PBCs and normal breast tissue were significantly higher (median r = 0.42) than the remaining loci (median r = 0.34) (Wilcoxon test, P &amp;lt; 2.2 x10-16), suggesting that methylation variations controlled by genetic variations exhibited a robust correlation between PBC and normal breast tissue DNA. In summary, our results suggest that the methylation status of CpG sites with large inter-person variations or genetically-controlled variations in PBC DNA correlate well with those in normal breast tissue. This supports the conduct of a genome-wide methylation study for breast cancer using PBC DNA. Citation Format: Xingyi Guo, Xiao-Ou Shu, Chun Li, Jirong Long, Bingshan Li, Yu-Tang Gao, Wei Zheng, Qiuyin Cai. Correlation of DNA methylation pattern between peripheral blood cell and normal breast tissue. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 1068. doi:10.1158/1538-7445.AM2015-1068

Abstract 1058: Novel insights into the genetic and epigenetic regulation of the MLH1 CpG island and shore in colorectal cancer

Abstract Gene silencing via CpG island hypermethylation contributes to colorectal cancer (CRC). We previously demonstrated that genetic variants (single nucleotide polymorphisms, or SNPs) in the MLH1 gene promoter region are associated with MLH1 CpG island hypermethylation, MLH1 protein loss, and DNA mismatch repair deficiency in tumors. Recently, CpG-rich “shore” regions were identified flanking many CpG islands which exhibiting distinct methylation profiles among different tissues and in normal versus tumor states. To explore the role of MLH1 CpG island shore methylation, we performed global methylation profiling (Illumina 450K microarrays) to investigate DNA methylation in peripheral blood cell (PBC) DNA of over 800 CRC cases and 800 controls (Savio et al., 2012). We found that MLH1 CpG island shore hypomethylation occurred in PBC DNA of both cases and controls. Intriguingly, individuals carrying variant alleles of the MLH1 promoter SNP rs1800734 incur significant hypomethylation at the MLH1 CpG island shore, located 1.5 kb upstream irrespective of disease status. Based on these observations we sought to determine whether these SNP-associated epigenetic changes were apparent in other tissues, including CRC and normal colonic tissue. To address this, we investigated MLH1 CpG island shore methylation in matched PBC, normal colon, and tumor DNA of 349 CRC cases using the real-time PCR-based technique, MethyLight. Methylation was significantly lower in PBC DNA of CRC cases with variant SNP genotypes (both heterozygous and homozygous variant carriers) and this association was also observed in normal colonic DNA. The association between genotype and epigenotype was lost in tumor DNA, as no differences in methylation were seen among different genotypes. Hypermethylation in tumor DNA compared to normal tissues (PBC and colon) was also observed. This hypermethylation and lack of association between SNP genotype and shore methylation in tumors may indicate an epigenetic switch occurring. Bisulfite sequencing of DNA from fresh frozen tumors and paired normal colonic mucosa have recapitulated these results, with hypomethylation incurred in variant SNP carriers. Hypermethylation in tumor compared to normal DNA was also observed. Bisulfite sequencing of four CRC cell lines has also revealed SNP-dependent hypermethylation at the MLH1 CpG island shore. Taken together, we have integrated methylation data from a microarray platform, sensitive real-time PCR, and bisulfite sequencing for a comprehensive and systematic analysis of DNA methylation at the CpG island shore of MLH1 in multiple tissues of CRC cases, controls, and cell lines. These results indicate that static genetic variants can modulate dynamic epigenetic regulation at the MLH1 gene region, and may play a role in colorectal tumorigenesis. Citation Format: Andrea J. Savio, Mathieu Lemire, Miralem Mrkonjic, Steven Gallinger, Brent W. Zanke, Thomas J. Hudson, Bharati Bapat. Novel insights into the genetic and epigenetic regulation of the MLH1 CpG island and shore in colorectal cancer. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 1058. doi:10.1158/1538-7445.AM2015-1058

Abstract 4596: Genetic variants associated with longer telomere length are associated with increased lung cancer risk among never-smoking women in Asia: A report from the Female Lung Cancer Consortium in Asia

Abstract Recent evidence from several relatively small nested case-control studies in prospective cohorts shows an association between longer telomere length measured phenotypically in peripheral white blood cell (WBC) DNA and increased lung cancer risk. We sought to further explore this relationship by examining a panel of 7 telomere-length associated genetic variants in a large study of 5,457 never-smoking female Asian lung cancer cases and 4,493 never-smoking female Asian controls using data from a previously reported genome-wide association study. Using a group of 1,536 individuals with phenotypically measured telomere length in WBCs in the prospective Shanghai Women's Health study, we demonstrated the utility of a genetic risk score (GRS) of 7 telomere-length associated variants to predict telomere length in an Asian population. We then found that GRSs used as instrumental variables to predict longer telomere length were associated with increased lung cancer risk (OR = 1.51 (95% CI = 1.34-1.69) for upper vs. lower quartile of the weighted GRS, P-value = 4.54×10−14) even after removing rs2736100 (P-value = 4.81×10−3), a SNP in the TERT locus robustly associated with lung cancer risk in prior association studies. Stratified analyses suggested the effect of the telomere-associated GRS is strongest among younger individuals. We found no difference in GRS effect between adenocarcinoma and squamous cell subtypes. Our results indicate that a genetic background that favors longer telomere length may increase lung cancer risk, which is consistent with earlier prospective studies relating longer telomere length with increased lung cancer risk. Citation Format: Mitchell J. Machiela, Chao A. Hsiung, Xiao-Ou Shu, Wei J. Seow, Zhaoming Wang, Keitaro Matsuo, Yun-Chul Hong, Adeline Seow, Chen Wu, H Dean Hosgood, Kexin Chen, Jiu-Cun Wang, Wanqing Wen, Tangchun Wu, Maria P. Wong, Yi-Long Wu, Pan-Chyr Yang, Baosen Zhou, Min-Ho Shin, Joseph F. Fraumeni, Wei Zheng, Dongxin Lin, Stephen J. Chanock, Nathaniel Rothman, Qing Lan. Genetic variants associated with longer telomere length are associated with increased lung cancer risk among never-smoking women in Asia: A report from the Female Lung Cancer Consortium in Asia. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 4596. doi:10.1158/1538-7445.AM2015-4596

Mitochondrial DNA Copy Number in Peripheral Blood Cells and Risk of Developing Breast Cancer.

Increased mitochondrial DNA (mtDNA) copy number in peripheral blood cells (PBC) has been associated with the risk of developing several tumor types. Here we evaluate sources of variation of this biomarker and its association with breast cancer risk in a prospective cohort study. mtDNA copy number was measured using quantitative real-time PCR on PBC DNA samples from participants in the UK-based Breakthrough Generations Study. Temporal and assay variation was evaluated in a serial study of 91 women, with two blood samples collected approximately 6-years apart. Then, associations with breast cancer risk factors and risk were evaluated in 1,108 cases and 1,099 controls using a nested case-control design. In the serial study, mtDNA copy number showed low assay variation but large temporal variation [assay intraclass correlation coefficient (ICC), 79.3%-87.9%; temporal ICC, 38.3%). Higher mtDNA copy number was significantly associated with younger age at blood collection, being premenopausal, having an older age at menopause, and never taking HRT, both in cases and controls. Based on measurements in a single blood sample taken on average 6 years before diagnosis, higher mtDNA copy number was associated with increased breast cancer risk [OR (95% CI) for highest versus lowest quartile, 1.37 (1.02-1.83); P trend = 0.007]. In conclusion, mtDNA copy number is associated with breast cancer risk and represents a promising biomarker for risk assessment. The relatively large temporal variation should be taken into account in future analyses.

Genetic variants associated with longer telomere length are associated with increased lung cancer risk among never-smoking women in Asia: a report from the female lung cancer consortium in Asia.

Recent evidence from several relatively small nested case-control studies in prospective cohorts shows an association between longer telomere length measured phenotypically in peripheral white blood cell (WBC) DNA and increased lung cancer risk. We sought to further explore this relationship by examining a panel of seven telomere-length associated genetic variants in a large study of 5,457 never-smoking female Asian lung cancer cases and 4,493 never-smoking female Asian controls using data from a previously reported genome-wide association study. Using a group of 1,536 individuals with phenotypically measured telomere length in WBCs in the prospective Shanghai Women's Health study, we demonstrated the utility of a genetic risk score (GRS) of seven telomere-length associated variants to predict telomere length in an Asian population. We then found that GRSs used as instrumental variables to predict longer telomere length were associated with increased lung cancer risk (OR = 1.51 (95% CI = 1.34-1.69) for upper vs. lower quartile of the weighted GRS, p value = 4.54 × 10(-14) ) even after removing rs2736100 (p value = 4.81 × 10(-3) ), a SNP in the TERT locus robustly associated with lung cancer risk in prior association studies. Stratified analyses suggested the effect of the telomere-associated GRS is strongest among younger individuals. We found no difference in GRS effect between adenocarcinoma and squamous cell subtypes. Our results indicate that a genetic background that favors longer telomere length may increase lung cancer risk, which is consistent with earlier prospective studies relating longer telomere length with increased lung cancer risk.

Clonal Hematopoiesis and Blood-Cancer Risk Inferred from Blood DNA Sequence

BackgroundCancers arise from multiple acquired mutations, which presumably occur over many years. Early stages in cancer development might be present years before cancers become clinically apparent.MethodsWe analyzed data from whole-exome sequencing of DNA in peripheral-blood cells from 12,380 persons, unselected for cancer or hematologic phenotypes. We identified somatic mutations on the basis of unusual allelic fractions. We used data from Swedish national patient registers to follow health outcomes for 2 to 7 years after DNA sampling.ResultsClonal hematopoiesis with somatic mutations was observed in 10% of persons older than 65 years of age but in only 1% of those younger than 50 years of age. Detectable clonal expansions most frequently involved somatic mutations in three genes (DNMT3A, ASXL1, and TET2) that have previously been implicated in hematologic cancers. Clonal hematopoiesis was a strong risk factor for subsequent hematologic cancer (hazard ratio, 12.9; 95% confidence interval, 5.8 to 28.7). Approximately 42% of hematologic cancers in this cohort arose in persons who had clonality at the time of DNA sampling, more than 6 months before a first diagnosis of cancer. Analysis of bone marrow–biopsy specimens obtained from two patients at the time of diagnosis of acute myeloid leukemia revealed that their cancers arose from the earlier clones.ConclusionsClonal hematopoiesis with somatic mutations is readily detected by means of DNA sequencing, is increasingly common as people age, and is associated with increased risks of hematologic cancer and death. A subset of the genes that are mutated in patients with myeloid cancers is frequently mutated in apparently healthy persons; these mutations may represent characteristic early events in the development of hematologic cancers. (Funded by the National Human Genome Research Institute and others.)

Clonal Hematopoiesis with Somatic Mutations Is a Common, Age-Related Condition Associated with Adverse Outcomes

Hematological malignancies are associated with recurrent somatic mutations in specific genes, and may be preceded by a pre-malignant state in which only the initial driver mutation(s) are present. For example, monoclonal gammopathy of unknown significance often precedes multiple myeloma and monoclonal B-lymphocytosis can precede chronic lymphocytic leukemia. Recent sequencing studies have identified genes that are recurrently mutated in acute myeloid leukemia, myelodysplastic syndromes, myeloproliferative neoplasms, acute lymphoblastic leukemia, and other hematological neoplasms. We hypothesized that a pre-malignant state comprised of a clonal expansion of cells harboring some of these recurrent mutations would be detectable in the blood of elderly individuals not known to have hematological disorders.To address this question, we analyzed whole exome sequencing data from peripheral blood cell DNA of 17,182 individuals. Most of these were sequenced for type 2 diabetes genetic association studies and were therefore unselected for hematological phenotypes. We looked for candidate somatic variants by identifying previously characterized single nucleotide variants (SNVs) and small insertions/deletions (indels) in 160 genes recurrently mutated in hematological malignancies. The presence of these variants was analyzed for association to hematological phenotypes, survival, and cardiovascular events.We identified a total of 805 candidate somatic variants (hereafter referred to as mutations) from 746 individuals in 73 genes. Somatic mutations were rarely detected in individuals younger than 40, but rose appreciably with age (Figure 1). At ages 70-79, 80-89, and 90-108 these clonal mutations were observed in 9.6% (220 out of 2299), 11.7% (37 out of 317), and 18.4% (19 out of 103) of individuals, respectively. The majority of the variants occurred in 3 genes: DNMT3A (403 variants), TET2 (72 variants), and ASXL1 (62 variants). The median variant allele fraction for the detected somatic mutations was 0.09, from which we infer that the pathologic clone represents on average 18% of circulating white blood cells.Clinical outcome data was available on a subset of subjects, with a median follow-up period of 8 years. Carrying a somatic mutation was associated with increased risk of developing a hematological malignancy (hazard ratio [HR] 11, 95% confidence interval [95% CI] 3.9-33 by competing risks regression). Harboring a mutation was also associated with an increase in all-cause mortality that could not be explained by death due to hematological malignancies alone (HR 1.4, 95% CI 1.1-1.8 by Cox proportional hazards model). We further found that these mutations were associated with type 2 diabetes (odds ratio 1.3, 95% CI 1.1-1.5) and increased risk of incident coronary heart disease (HR 2.0, 95% CI 1.2-3.4) and ischemic stroke (HR 2.6, 95% CI 1.4-4.8) in multivariable regression models.We conclude that clonal hematopoiesis associated with a somatic mutation in a known cancer-causing gene is a common pre-malignant condition in the elderly. This entity is associated with increased risk of transformation to hematological malignancy, as well as increased all-cause mortality, possibly due to increased cardio-metabolic disease. While the link between somatic mutations and cancer is well established, the relationship between clonal hematopoiesis and cardio-metabolic disease requires further study.Figure 1 [Display omitted] DisclosuresGetz:The Broad Institute, Inc.: PCT/US2013/057128​ (Detecting Variants in Sequencing Data and Benchmarking Methods and Apparatus for Analyzing and Quantifying DNA Alterations in Cancer)​ Patent pending Patents & Royalties; Appistry: Certain NGS analysis tools of Broad Institute are made available for commercial use via Appistry, Certain NGS analysis tools of Broad Institute are made available for commercial use via Appistry Other. Ebert:Genoptix: Consultancy, Patents & Royalties; Celgene: Consultancy, Research Funding.

Subcutaneous bortezomib is highly effective for pure red cell aplasia after ABO‐incompatible haematopoietic stem cell transplantation

Subcutaneous bortezomib is highly effective for pure red cell aplasia after <scp>ABO</scp>‐incompatible haematopoietic stem cell transplantation

Evaluation of protein expression and DNA methylation profiles detected by pyrosequencing in invasive breast cancer

Breast carcinoma is the most common cancer with high mortality caused by metastatic disease. New molecular biomarkers predicting the tumour's metastatic potential would therefore improve metastasis prevention and personalised care. The aim of the study was to investigate the relationship between DNA methylation levels in invasivity and metastasising associated genes with aberrant protein expression and also to evaluate whether a similar DNA methylation level is present in the tumour and circulating cell-free DNA for utilising plasma DNA methylation as prognostic biomarker. By using pyrosequencing, we analysed DNA methylation levels of 11 genes, namely APC, ADAM23, CXCL12, ESR1, PGR B, CDH1, RASSF1A, SYK, TIMP3, BRMS1 and SOCS1 in tumour, plasma and peripheral blood cells from 34 patients with primary breast cancer, as well as plasma and peripheral blood cells from 50 healthy controls. Simultaneously, the expression of related proteins in paraffin-embedded tumour samples was evaluated by immunohistochemistry. Statistical analysis was performed by SPSS statistics 15.0 software. Tumour DNA hypermethylation was found in most commonly methylated RASSF1A (71.9%), APC (55.9%), ADAM23 (38%) and CXCL12 (34.4%) genes with methylation levels up to 86, 86, 53 and 64 %, respectively. In tumours, significantly higher methylation levels were found in nine genes, compared with the patients´ peripheral blood cell DNA. Furthermore, in patients methylation levels in peripheral blood cell DNA were significantly higher than in controls in CXCL12, ESR1 and TIMP3 genes, but the values did not exceed 15%. On the other hand, no correlations were observed in patients between DNA methylation in tumours and cell-free plasma DNA. Moreover, in patients and controls nearly identical values of cumulative DNA methylation (43.6 % ± 20.1 vs. 43.7 % ± 15.0) were observed in plasma samples. A variable spectrum from high to none expressions presented in tumour tissues in all of the proteins evaluated, however in APC and CXCL12 genes a visible decreasing trend of mean DNA methylation level with increasing expression of the corresponding protein was observed. The DNA methylation profiles manifested in our group of breast carcinomas are cancer specific, but they are not the only cause that affects the silencing of evaluated genes and the decrease of relevant protein products. The clinical utility of DNA methylation testing in peripheral blood cell DNA for cancer diagnosis and therapy need to be further investigated.

Whole Exome Sequencing and Analysis Of Mutations In Sézary Syndrome

Introduction and RationaleSézary Syndrome (SS), the leukemic variant of cutaneous T-cell lymphoma CTCL), is characterized by generalized erythroderma and >1000 circulating central memory T-cells with a CD4+CD26- phenotype, Other clinical features include reactive adenopathy, keratoderma, ectropion, pruritus, and colonization with Staphylococcus aureus. SS can arise de novo or emerge in patients with Mycosis Fungoides (MF). Morphologically, SS cells were first recognized by their convoluted nuclei and karyotype analysis has shown the presence of multiple chromosomal abnormalities and copy number variants, none of which are disease defining; however, the mutational landscape of SS has not been investigated systematically. Here we present initial findings from profiling somatic mutation by whole exome sequencing. MethodsIn order to delineate the genomic mutation profile of Sézary cells we compared protein coding sequences from CD4+ and CD4- peripheral blood cell DNA to that from autologous cultured fibroblasts derived from ten newly diagnosed SS patients with high percentages of circulating SS cells. CD4+ T-cells were selected by a Miltenyi Biotec CD4+ T Cell Isolation Kit and genomic DNA was extracted from CD4+ T-cells, residual CD4- cells, and autologous fibroblasts from each patient using an All Prep DNA/RNA/Protein Mini Kit (Qiagen). Purity of CD4+ T-cell sorting confirmed by flow cytometry was > 90% purity. Skin biopsies from SS non-lesional skin were explanted, cultured for 5-6 weeks, and used to extract genomic control DNA for each patient. Whole exome DNA was enriched by capture using a Nimblegen VCRome version 2.1 array. Nine billion bases of sequence was generated for each sample on an Illumina HiSeq 2000 run for 101 cycles. This resulted in approximately 95% target covered to a minimum depth of 20 bases, with and overall average coverage of over 130 bases in all samples. Somatic mutations were called by comparing sequence from all tissues to the human reference sequence (GRC37) and then subtracting nucleotide variation found in the fibroblast lines from variation found in the CD4- and CD4+ cell populations. ResultsWe found an average of 82 mutations per patient in the CD4+ cells. TP53 with 4 mutations was the most commonly mutated, three patients harbored MSH3 mutations, which may contribute to higher mutation frequencies in those patients. Singleton mutations in MYC, MLLT4 are among cancer-related transcription factors, and singleton NF1 and Notch4 suggest aberrant signaling. Surprisingly, the CD4- cells also harbored TP53 mutations in 3 of the 4 patients. ConclusionsWhole exome sequencing revealed recurrent mutations in two genes: TP53 and MSH3 and hinted at involvement of RAS and Notch pathway signaling, although no RAS mutations were observed. The presence of TP53 mutation in CD4- cells was unanticipated, and may result from mutation of a common progenitor cell population in the CD4- cells. Studies are underway, including analysis of copy number gains and losses to further elucidate the mutational patterns in SS patients. Disclosures:No relevant conflicts of interest to declare.

Overtraining is associated with DNA damage in blood and skeletal muscle cells of Swiss mice.

BackgroundThe alkaline version of the single-cell gel (comet) assay is a useful method for quantifying DNA damage. Although some studies on chronic and acute effects of exercise on DNA damage measured by the comet assay have been performed, it is unknown if an aerobic training protocol with intensity, volume, and load clearly defined will improve performance without leading to peripheral blood cell DNA damage. In addition, the effects of overtraining on DNA damage are unknown. Therefore, this study aimed to examine the effects of aerobic training and overtraining on DNA damage in peripheral blood and skeletal muscle cells in Swiss mice. To examine possible changes in these parameters with oxidative stress, we measured reduced glutathione (GSH) levels in total blood, and GSH levels and lipid peroxidation in muscle samples.ResultsPerformance evaluations (i.e., incremental load and exhaustive tests) showed significant intra and inter-group differences. The overtrained (OTR) group showed a significant increase in the percentage of DNA in the tail compared with the control (C) and trained (TR) groups. GSH levels were significantly lower in the OTR group than in the C and TR groups. The OTR group had significantly higher lipid peroxidation levels compared with the C and TR groups.ConclusionsAerobic and anaerobic performance parameters can be improved in training at maximal lactate steady state during 8 weeks without leading to DNA damage in peripheral blood and skeletal muscle cells or to oxidative stress in skeletal muscle cells. However, overtraining induced by downhill running training sessions is associated with DNA damage in peripheral blood and skeletal muscle cells, and with oxidative stress in skeletal muscle cells and total blood.

Lack of correlation between the size of HIV proviral DNA reservoir and the level of immune activation in HIV-infected patients with a sustained undetectable HIV viral load for 10 years

BackgroundThe persistence of HIV residual replication in patients with an undetectable plasma viral load (pVL) may limit immune recovery and facilitate inflammation-induced comorbidities. ObjectiveThe objective was to evaluate any correlation between immune restoration and intracellular [IC] HIV-DNA in cART-treated patients with a sustained undetectable pVL. Study designThis retrospective cross sectional study included 62 patients with a median duration of undetectable pVL of 10.3 years. IC HIV DNA in peripheral mononuclear blood cells (PBMCs) and T cell subsets were measured at the last visit. pVL, CD4+ and CD8+ T cell counts were retrospectively collected from the onset of long-term inhibition by antiretroviral treatment. The patients were separated into two groups: 27 non-blippers (sustained pVL<threshold value during all the visits) and 35 blippers (≥1 episodes of pVL>threshold but <1000 copies/ml). The median pVL in blippers was 115 copies/ml. ResultsThe median IC HIV DNA rate was 34 copies/106 PBMCs (71% ≥20 copies/106 PBMCs) with no significant difference between the groups. The proportion of CD8+CD38+ and CD8+DR+ T cells was higher in blipper patients, but the difference was only significant for the CD8+DR+ marker (p=0.036). No correlation was found between markers of immune activation on CD4+ and CD8+ T cells and the IC HIV-DNA level. ConclusionNo relation was found between the size of HIV reservoirs and immune activation in patients with sustained undetectable pVL. Mechanisms of immune activation have to be better understood in order to define specific therapeutic interventions.

Longer Telomere Length in Peripheral White Blood Cells Is Associated with Risk of Lung Cancer and the rs2736100 (CLPTM1L-TERT) Polymorphism in a Prospective Cohort Study among Women in China

A recent genome-wide association study of lung cancer among never-smoking females in Asia demonstrated that the rs2736100 polymorphism in the TERT-CLPTM1L locus on chromosome 5p15.33 was strongly and significantly associated with risk of adenocarcinoma of the lung. The telomerase gene TERT is a reverse transcriptase that is critical for telomere replication and stabilization by controlling telomere length. We previously found that longer telomere length measured in peripheral white blood cell DNA was associated with increased risk of lung cancer in a prospective cohort study of smoking males in Finland. To follow up on this finding, we carried out a nested case-control study of 215 female lung cancer cases and 215 female controls, 94% of whom were never-smokers, in the prospective Shanghai Women’s Health Study cohort. There was a dose-response relationship between tertiles of telomere length and risk of lung cancer (odds ratio (OR), 95% confidence interval [CI]: 1.0, 1.4 [0.8–2.5], and 2.2 [1.2–4.0], respectively; P trend = 0.003). Further, the association was unchanged by the length of time from blood collection to case diagnosis. In addition, the rs2736100 G allele, which we previously have shown to be associated with risk of lung cancer in this cohort, was significantly associated with longer telomere length in these same study subjects (P trend = 0.030). Our findings suggest that individuals with longer telomere length in peripheral white blood cells may have an increased risk of lung cancer, but require replication in additional prospective cohorts and populations.

Comprehensive DNA methylation analysis of peripheral blood cells derived from patients with first-episode schizophrenia

Epidemiological studies have revealed that schizophrenia is highly heritable. However, genetic studies have not fully elucidated its etiology. Accumulating evidence suggests that epigenetic alterations may provide an additional explanation of its pathophysiology. We investigated the methylation profiles of DNA in peripheral blood cells from 18 patients with first-episode schizophrenia (FESZ) and from 15 normal controls. Schizophrenia patients were confined to those at the stage of first-episode psychosis. We analyzed the DNA methylation status of 27,578 CpG sites by means of the Illumina Infinium HumanMethylation27 BeadChip array. Differentially methylated CpG sites, which were particularly abundant within CpG islands, were enriched in genes related to the nuclear lumen, to transcription factor binding, and to nucleotide binding. We also observed differential methylation of the promoters of HTR1E and COMTD1, which are functionally related to genes found to be differentially methylated in schizophrenia patients in previous studies. Our results indicate the site-specific epigenetic alterations in patients with FESZ.

MLH1 region polymorphisms show a significant association with CpG island shore methylation in a large cohort of healthy individuals.

Single nucleotide polymorphisms (SNPs) are the most common form of genetic variation. We previously demonstrated that SNPs (rs1800734, rs749072, and rs13098279) in the MLH1 gene region are associated with MLH1 promoter island methylation, loss of MLH1 protein expression, and microsatellite instability (MSI) in colorectal cancer (CRC) patients. Recent studies have identified less CpG-dense “shore” regions flanking many CpG islands. These shores often exhibit distinct methylation profiles between different tissues and matched normal versus tumor cells of patients. To date, most epigenetic studies have focused on somatic methylation events occurring within solid tumors; less is known of the contributions of peripheral blood cell (PBC) methylation to processes such as aging and tumorigenesis. To address whether MLH1 methylation in PBCs is correlated with tumorigenesis we utilized the Illumina 450 K microarrays to measure methylation in PBC DNA of 846 healthy controls and 252 CRC patients from Ontario, Canada. Analysis of a region of chromosome 3p21 spanning the MLH1 locus in healthy controls revealed that a CpG island shore 1 kb upstream of the MLH1 gene exhibits different methylation profiles when stratified by SNP genotypes (rs1800734, rs749072, and rs13098279). Individuals with wild-type genotypes incur significantly higher PBC shore methylation than heterozygous or homozygous variant carriers (p<1.1×10−6; ANOVA). This trend is also seen in CRC cases (p<0.096; ANOVA). Shore methylation also decreases significantly with increasing age in cases and controls. This is the first study of its kind to integrate PBC methylation at a CpG island shore with SNP genotype status in CRC cases and controls. These results indicate that CpG island shore methylation in PBCs may be influenced by genotype as well as the normal aging process.

Demethylation of the aryl hydrocarbon receptor repressor as a biomarker for nascent smokers

Epigenetic modifications to peripheral white blood cell DNA occur in response to a wide variety of exposures. In prior work, we and others have shown that broad changes in DNA methylation, particularly at the aryl hydrocarbon receptor repressor (AHRR) locus, occur in samples from subjects with long histories of smoking. However, given the large number of epigenetic changes that occur in response to prolonged smoking, the primacy of the response at AHRR and the sensitivity of these changes to low levels of smoking are not known. Therefore, we examined the association of smoking to genome lymphocyte DNA methylation status in a representative sample of 399 African American youths living in the rural South that includes 72 subjects with less than one half-pack year of exposure. Consistent with our prior findings, we found a stepwise effect of smoking on DNA methylation among youth with relatively brief exposure histories at a CpG residue in AHRR (cg05575921) (FDR corrected p values; 3 × 10−7 and 0.09 in the male and female samples, respectively) that was identified in previous studies and at which the effects of smoking were significant, even in those subjects with less than one half pack year exposure. We conclude that AHRR demethylation at cg05575921 in peripheral cells may serve as an early, sensitive biomarker for even low levels of exposure to tobacco smoke, providing a non-self-report alternative for nascent exposure to tobacco smoke. We also suggest that the AHRR/AHR pathway may be functional in the response of peripheral white blood cells to tobacco smoke exposure.

Plasma estrone sulfate concentrations and genetic variation at the CYP19A1 locus in postmenopausal women with early breast cancer treated with letrozole

Estrogen synthesis suppression induced by aromatase inhibitors in breast cancer (BC) patients may be affected by single nucleotide polymorphisms (SNPs) of the gene encoding aromatase enzyme, CYP19A1. We assessed the association between plasma estrone sulfate (ES), letrozole treatment, and four SNPs of CYP19A1 gene (rs10046 C>T, rs4646 G>T, rs749292 C>T, rs727479 T>G) which seem to be related to circulating estrogen levels. Patients were enrolled into a prospective, Italian multi-center clinical trial (Gruppo Italiano Mammella, GIM-5) testing the association of CYP19A1 SNPs with the efficacy of letrozole adjuvant therapy, in postmenopausal early BC patients. SNPs were identified from peripheral blood cell DNA. Plasma ES concentrations were evaluated by Radio Immuno Assay. Blood samples were obtained immediately before letrozole therapy (N = 204), at 6-weeks (N = 178), 6 (N = 152) and 12-months (N = 136) during treatment. Medians (IQR) of ES were 160 pg/mL (85–274) at baseline, 35 pg/mL (12–64) at 6-weeks, 29 pg/mL (17–48) at 6 months and 25 pg/mL (8–46) after 12 months treatment. No statistically significant association was evident between polymorphisms and ES circulating levels during letrozole therapy. Letrozole suppression of the aromatase enzyme function is not affected by polymorphisms of CYP19A1 gene in postmenopausal BC patients.