Pilot Study of Markers for High-grade Anal Dysplasia in a Southern Cohort From the Women's Interagency Human Immunodeficiency Virus Study.

BACKGROUND: Anal cancer rates have increased, particularly in human immunodeficiency virus (HIV)-infected (HIV+) women. We assessed factors associated with anal precancer in HIV+ and at-risk HIV-negative women from the Atlanta Women's Interagency HIV Study cohort. METHODS: All participants underwent high-resolution anoscopy and anal cytology and had anal and cervical samples collected. Specimens were tested for 37 human papillomavirus (HPV) types and for FAM19A4 and microRNA124-2 promoter methylation. Binary logistic regression and multivariate analysis were conducted with histologic anal high-grade squamous intraepithelial lesion (A-HSIL) as the dependent variable. RESULTS: Seventy-five women were enrolled: 52 (69%) were HIV+ with three-fourths having undetectable viral load; 64 (86%) were black; mean age was 49 ± 8 years. Forty-nine (65%) anal cytology samples were abnormal, and 38 (51%) of anal samples were positive for at least 1 of 13 high-risk HPV (hrHPV) types. Thirteen (18%) anal biopsies identified A-HSIL. Hypermethylation of FAM19A4 and/or microRNA124-2 was found in 69 (95%) anal samples and 19 (26%) cervical samples. In multivariate analyses, the odds of having A-HSIL were >6 times higher in women with anal hrHPV (adjusted odds ratio [aOR], 6.08 [95% confidence interval {CI}, 1.27-29.18], P = .02) and with positive cervical methylation (aOR, 6.49 [95% CI, 1.66-25.35], P = .007), but not significantly higher in women with positive anal methylation. CONCLUSIONS: Anal hrHPV and promoter hypermethylation in the cervix show promise as biomarkers for anal cancer screening in HIV+ and at-risk HIV-negative women. Greater understanding of gene silencing by promoter hypermethylation in anal carcinogenesis is needed.

Simultaneous extraction of mRNA and microRNA from whole blood stabilized in tempus tubes.

OBJECTIVE: Studies of mRNA and miRNA expression profiling increasingly use stabilized whole blood. Commercial RNA extraction kits do not provide information about the simultaneous recovery of both mRNA and miRNA. This study evaluated yield, quality, integrity and representation of mRNA and miRNA from whole blood stabilized in Tempus tubes using three RNA extraction kits; two filter-based (Tempus and Norgen) and one bead-based (MagMax; manual vs. semi-automated, and with and without DNase treatment). RESULTS: All RNA extraction kits and methods resulted in similar yields of mRNA (total RNA yield, quality, integrity and representation) whereas there were differences in yields of miRNA. MagMax, either manual or semi-automated, with or without DNase treatment, yielded 1.6-2.2-fold more miRNA than Tempus and Norgen kits. In addition, MagMax and Norgen methods gave greater than 12-fold more and 3.3-fold less enrichment of specific miRNA targets, respectively, in comparison to Tempus extraction reagents. This study identified MagMax kit for simultaneous recovery of both mRNA and miRNA from whole blood collected in Tempus tubes.

DNA Methylation Signature of Post-injury Neointimal Cells During Vascular Remodeling in the Rat Balloon Injury Model.

Vascular smooth muscle cell (VSMC) accumulation in the neointimal is a common feature in vascular diseases such as atherosclerosis, transplant arteriosclerosis and restenosis. In this study, we isolated the neointimal cells and uninjured residential vascular smooth muscle cells by laser micro dissection and carried out single-cell whole-genome methylation sequencing. We also sequenced the bisulfite converted genome of circulating bone-marrow-derived cells such as peripheral blood mononuclear cells (PBMC) and bone marrow mononuclear cells (BMMC). We found totally 2,360 differential methylation sites (DMS) annotated to 1,127 gene regions. The majority of differentially methylated regions (DMRs) were located in intergenic regions, outside those CpG islands and island shores. Interestingly, exons have less DMRs than promotors and introns, and CpG islands contain more DMRs than islands shores. Pearson correlation analysis showed a clear clustering of neointimal cells with PBMC/BMMC. Gene set enrichment analysis of differentially methylated CpG sites revealed that many genes were important for regulation of VSMC differentiation and stem cell maintenance. In conclusion, our results showed that neointimal cells are more similar to the progenitor cells in methylation profile than the residential VSMCs at the 30th day after the vascular injury.

Presence of multimeric isoforms of human C-reactive protein in tissues and blood.

The baseline concentration of C-reactive protein (CRP) has been associated with a wide array of human diseases. In epidemiological studies and in the clinic, CRP is typically measured as a pentamer, composed of 5 identical CRP subunits. The present study aimed to determine whether other isoforms were present in the blood by examining CRP conformations. Transgenic rats expressing human CRP under the mouse albumin promoter were generated and genotyped. Non­reducing western blotting was performed using the blood and tissues of transgenic rats and human patients. CRP concentrations in human blood were examined by enzyme­linked immunosorbent assay. In addition to the pentameric isoform, CRP was detected as a trimer and tetramer in the blood of human CRP transgenic rats. Furthermore, trimeric and tetrameric CRP was observed in various tissues, including aorta, liver, kidney, pancreas, heart and skeletal muscle. Notably, these two isoforms appeared to be age­associated, as they were detected only in the blood and tissues of older transgenic rats. The existence of additional CRP isoforms was confirmed in the blood of human patients by non­reducing western blotting. Clinical and epidemiological studies typically focus on CRP concentration. However, the results of the present study suggest that, in addition to concentration, CRP conformation may require analysis.

High-accuracy haplotype imputation using unphased genotype data as the references.

Enormously growing genomic datasets present a new challenge on missing data imputation, a notoriously resource-demanding task. Haplotype imputation requires ethnicity-matched references. However, to date, haplotype references are not available for the majority of populations in the world. We explored to use existing unphased genotype datasets as references; if it succeeds, it will cover almost all of the populations in the world. The results showed that our HiFi software successfully yields 99.43% accuracy with unphased genotype references. Our method provides a cost-effective solution to breakthrough the bottleneck of limited reference availability for haplotype imputation in the big data era.

Functional pseudogenes inhibit the superoxide production.

We recently discovered a dynamic copy number variation on the NCF1 (neutrophil cytosolic factor 1) pseudogenes in human populations. In this study, we investigated whether these pseudogenes are functional or junk as described before. We sequenced the RNAs transcribed from the genome of this locus, and discovered over 10 splicing isoforms from the NCF1 pseudogenes. We cloned 4 splicing isoforms into expression vectors and introduced them into human vascular endothelial cells by transient transfection. We then used two chemical approaches to measure the superoxide production in the cells with and without these pseudogene overexpression. Our data showed that three pseudogene splicing products remarkably reduced the superoxide production after the GFP (Green fluorescent protein) normalization. We used an anti-HA (Hemagglutinin A) tag antibody to stain the cells and confirmed that the proteins transcribed from the NCF1 pseudogene were exclusively localized in the cytoplasm in the perinuclear area in the transient transfection assays. We further examined the tissue distribution of these splicing isoforms of NCF1 pseudogenes in human tissues by quantitative real-time PCR analysis. Our data showed that although these splicing variants are ubiquitously expressed in non-immune tissues in human, they seem to be under a tight control of transcription regulation and show a non-random distribution pattern across tissues. This study challenges the concept that pseudogenes in human genome are only junks without biological functions. Moreover, it suggests that those pseudogenes in human genome may serve as a natural resource for novel drug discovery.

A preliminary exploration on DNA methylation of transgene across generations in transgenic rats.

Epigenetic heritability is an important issue in the field of genetics and also in the development of many human diseases. In this study, we created a transgenic rat model and investigated the transgenerational methylation patterns in these animals. The transgene DNA fragment was unmethylated before it was injected into the pronucleus, so it is a good model to study the inheritance of DNA methylation patterns. We performed bisulfite sequencing on 23 CpG dinucleotides on the transgene across three generations in two tissues. We observed that the transgene was heavily methylated in the liver (87.53%) from the founder generation, whereas its methylation rate was much lower in the kidney (70.47%). Spearman correlation analysis showed that there was a strong correlation on the methylation status between different generations in the same tissue, which was observed in both liver and kidney, and among all individuals in this pedigree. This study provided some evidence that DNA methylation patterns acquired in the founder animal can be passed to the offspring.

The mirror RNA expression pattern in human tissues.

It has been realized in recent years that non-coding RNAs are playing important roles in genome functions and human diseases. Here we developed a new technology and observed a new pattern of gene expression. We observed that over 72% of RNAs in human genome are expressed in forward-reverse pairs, just like mirror images of each other between forward expression and reverse expression; the overview showed that it cannot be simply described as transcript overlapping, so we designated it as mirror expression. Furthermore, we found that the mirror expression is gene-specific and tissue-specific, and less common in the proximal promoter regions. The size of the shadows varies between different genes, different tissues and different classes. The shadow expression is most significant in the Alu element, it was also observed among L1, Simple Repeats and LTR elements, but rare in other repeats such as low-complexity, LINE/L2, DNA and MIRs. Although there is no evidence yet about the relationship of this mirror pattern and double-strand RNA (dsRNA), this new striking pattern provides a new clue and a new direction to unveil the role of RNAs in the genome functions and diseases.

A method to evaluate genome-wide methylation in archival formalin-fixed, paraffin-embedded ovarian epithelial cells.

BACKGROUND: The use of DNA from archival formalin and paraffin embedded (FFPE) tissue for genetic and epigenetic analyses may be problematic, since the DNA is often degraded and only limited amounts may be available. Thus, it is currently not known whether genome-wide methylation can be reliably assessed in DNA from archival FFPE tissue. METHODOLOGY/PRINCIPAL FINDINGS: Ovarian tissues, which were obtained and formalin-fixed and paraffin-embedded in either 1999 or 2011, were sectioned and stained with hematoxylin-eosin (H&E).Epithelial cells were captured by laser micro dissection, and their DNA subjected to whole genomic bisulfite conversion, whole genomic polymerase chain reaction (PCR) amplification, and purification. Sequencing and software analyses were performed to identify the extent of genomic methylation. We observed that 31.7% of sequence reads from the DNA in the 1999 archival FFPE tissue, and 70.6% of the reads from the 2011 sample, could be matched with the genome. Methylation rates of CpG on the Watson and Crick strands were 32.2% and 45.5%, respectively, in the 1999 sample, and 65.1% and 42.7% in the 2011 sample. CONCLUSIONS/SIGNIFICANCE: We have developed an efficient method that allows DNA methylation to be assessed in archival FFPE tissue samples.

A promoter that drives gene expression preferentially in male transgenic rats.

Gender-preferential gene expression is a widespread phenomenon in humans. It is important to study how gender differences influence the pathogenesis of various diseases and response to specific drugs. The aim of this study is to determine if the mouse albumin enhancer/promoter may serve as the promoter to introduce gender-preferential gene expression in transgenic animals. We created four independent transgenic rat lines in which the human C-reactive protein transgene was under the control of mouse albumin enhancer/promoter. Quantitative real time RT-PCR analysis showed that transgene expression in the liver of male rats was significantly higher than transgene expression in the female rats (P < 0.05).There was a 5.3-fold (male/female) difference in line-519, and a 12.2-fold (male/female) difference in line-488. Enzyme-linked immunosorbent assay showed that the serum of male transgenic rats had a 13- to 679-fold difference at the protein level on transgene production compared with female transgenic rats. The male-to-female difference in gene expression was 10- to 17-fold in the liver of transgenic rats. Orchiectomy dramatically reduced protein production from the transgene in the liver. Testosterone administration into female rats did not increase the transgene expression, but estrogen administration into the male rats reduced transgene expression. This study provides a valuable tool for investigating the pathological roles of genes that are expressed in a gender-preferential manner in human disease.

Multimeric stability of human C-reactive protein in archived specimens.

BACKGROUND: C-reactive protein (CRP) is a marker of inflammation and a risk predictor of cardiovascular disease. Current CRP assays are focused on the quantification of the CRP levels as pentamers. However, CRP can be present as other multimeric forms. There will be a market need to measure the CRP multimeric structure in addition to the levels in human populations. To meet this need, we investigated whether the long-term archived samples could be used instead of freshly collected samples. METHODOLOGY/PRINCIPAL FINDINGS: The specimens of serum, plasma and tissues were collected from transgenic rats expressing the human CRP. These samples were stored at 4°C, -20°C and -80°C for different periods. Non-denaturing Western blot analysis was used to observe the influence of storage conditions to multimeric structures of human CRP. Our results showed that there was no difference on multimeric structures of human CRP between samples stored at 4°C, -20°C and -80°C, between samples stored at -80°C for twenty-four hours and three months, and between plasma and serum. CONCLUSIONS/SIGNIFICANCE: This study implicated that archived samples stored at these conditions in those large longitudinal studies could be used for investigating the multimeric structures of CRP. Our report may speed up these researches and save labors and budget by enabling them to use currently available archived samples rather than freshly collected samples.

An inherited heteroplasmic mutation in mitochondrial gene COI in a patient with prostate cancer alters reactive oxygen, reactive nitrogen and proliferation.

Mitochondrial DNA (mtDNA) mutations have been found in many cancers but the physiological derangements caused by such mutations have remained elusive. Prostate cancer is associated with both inherited and somatic mutations in the cytochrome c oxidase (COI) gene. We present a prostate cancer patient-derived rare heteroplasmic mutation of this gene, part of mitochondrial respiratory complex IV. Functional studies indicate that this mutation leads to the simultaneous decrease in cytochrome oxidation, increase in reactive oxygen, and increased reactive nitrogen. These data suggest that mitochondrial DNA mutations resulting in increased reactive oxygen and reactive nitrogen generation may be involved in prostate cancer biology.

Mitochondrial DNA mutation stimulates prostate cancer growth in bone stromal environment.

BACKGROUND AND OBJECTIVES: Mitochondrial DNA (mtDNA) mutations, inherited and somatically acquired, are common in clinical prostate cancer. We have developed model systems designed to study specific mtDNA mutations in controlled experiments. Because prostate cancer frequently metastasizes to bone we tested the hypothesis that mtDNA mutations enhance prostate cancer growth and survival in the bone microenvironment. METHODS: The pathogenic nucleotide position (np) 8993 mDNA mutation was introduced into PC3 prostate cancer cells by cybrid formation. Wild-type and mutant cybrids were grown as nude mouse subcutaneous xenografts with or without bone stromal cell co-inoculation. Cybrids were also grown in the intratibial space. Tumor growth was assayed by direct tumor measurement and luciferase chemiluminescence. Gene expression was assayed using cDNA microarrays confirmed by real time PCR, western blot analysis and immunohistochemistry. RESULTS: Cybrids with the 8,993 mtDNA mutation grew faster than wild-type cybrids. Further growth acceleration was demonstrated in the bone microenvironment. A 37 gene molecular signature characterized the growth advantage conferred by the mtDNA mutation and bone microenvironment. Two genes of known importance in clinical prostate cancer, FGF1 and FAK, were found to be substantially upregulated only when both mtDNA mutation and bone stromal cell were present. CONCLUSIONS: The ATP6 np 8,993 mtDNA mutation confers a growth advantage to human prostate cancer that is most fully manifest in the bone microenvironment. The identification of specific molecular alterations associated with mtDNA mutation and growth in bone may allow new understanding of prostate cancer bone metastasis.