DHCPP

Chronic fatigue syndrome (CFS) is a debilitating illness lacking consistent anatomic lesions and eluding conventional laboratory diagnosis. Demonstration of the utility of the blood for gene expression profiling and biomarker discovery would have implications into the pathophysiology of CFS. The objective of this study was to determine if gene expression profiles of peripheral blood mononuclear cells (PMBCs) could distinguish between subjects with CFS and healthy controls. Total RNA from PBMCs of five CFS cases and seventeen controls was labeled and hybridized to 1764 genes on filter arrays. Gene intensity values were analyzed by various classification algorithms and nonparametric statistical methods. The classification algorithms grouped the majority of the CFS cases together, and distinguished them from the healthy controls. Eight genes were differentially expressed in both an age-matched case-control analysis and when comparing all CFS cases to all controls. Several of the diffrentially expressed genes are associated with immunologic functions (e.g., CMRF35 antigen, IL-8, HD protein) and implicate immune dysfunction in the pathophysiology of CFS. These results successfully demonstrate the utility of the blood for gene expression profiling to distinguish subjects with CFS from healthy controls and for identifying genes that could serve as CFS biomarkers.

Objectives. Polymorphisms in human papillomavirus (HPV) type 16 have been shown to be related to geographic areas and are broadly classified as European (E), African (Af), Asian (As), or Asian-American (AA). Certain variants have been reported as being more likely to cause cervical disease; our objectives were to identify new HPV16 polymorphisms, to determine the linkage of the E2 and E6/E7 regions and to determine the minimum sequence necessary to classify variants.

Acute infection is known to perturb psycho-neuroendocrine-immune (PNI) gene expression. Oligonucleotide microarrays were used to examine PNI gene expression in the peripheral blood of 13 subjects with infectious mononucleosis (IM). Novel peripheral blood gene expression activity was correlated with central-nervous-system-mediated symptoms including fatigue and sleep disturbance. Of note, expression of the MADS box transcription enhancer factor 2 polypeptide C (MEF2C) gene, previously implicated in skeletal muscle myogenesis, correlated with symptoms of musculo-skeletal pain and fatigue. Expression of the hypocretin/orexin receptor HCRTR2, which has been implicated in narcolepsy, correlated with sleep disturbance. And, VACHT, the vesicular acetylcholine transporter, was highly correlated with neurocognitive disturbance. The expression of both HCRTR2 and MEF2C in the peripheral blood was validated by reverse transcription PCR. Thus, investigation of the PNI response in peripheral blood may provide novel insights into the complex pathophysiology of centrally mediated disease states.

Before gene expression profiling with microarray technology can be transferred to the diagnostic setting, we must have alternative approaches for synthesizing probe from limited RNA samples, and we must understand the limits of reproducibility in interpreting gene expression results. The current gold standard of probes for use with both microarrays and high-density filter arrays are synthesized from 1 g of purified poly(A)؉ RNA. We evaluated two approaches for synthesizing cDNA probes from total RNA with subsequent hybridization to high-density filter arrays: 1) reverse transcription (RT) of 5 g total RNA and 2) RT-polymerase chain reaction (RT-PCR) of 1 g total RNA, using the SMART system. The reproducibility of these two approaches was compared to the current gold standard. All three methods were highly reproducible. Triplicate experiments resulted in the following concordance correlation coefficients to evaluate reproducibility: 0.88 for the gold standard, 0.86 for cDNA probe synthesized by RT from total RNA, and 0.96 for the SMART cDNA probe synthesized from total RNA. We also compared the expression profile of 588 genes for the total RNA methods to that obtained with the gold standard. Of 150 positive genes detected by the gold standard, 97 (65%) were detected by cDNA probe synthesized by RT of total RNA, and 122 (81%) were detected by the SMART cDNA probe. We conclude that SMART cDNA probe produces highly reproducible results and yields gene expression profiles that represent the majority of transcripts detected with the gold standard.

Chronic fatigue syndrome (CFS) is a debilitating illness lacking consistent anatomic lesions and eluding conventional laboratory diagnosis. Demonstration of the utility of the blood for gene expression profiling and biomarker discovery would have implications into the pathophysiology of CFS. The objective of this study was to determine if gene expression profiles of peripheral blood mononuclear cells (PMBCs) could distinguish between subjects with CFS and healthy controls. Total RNA from PBMCs of five CFS cases and seventeen controls was labeled and hybridized to 1764 genes on filter arrays. Gene intensity values were analyzed by various classification algorithms and nonparametric statistical methods. The classification algorithms grouped the majority of the CFS cases together, and distinguished them from the healthy controls. Eight genes were differentially expressed in both an age-matched case-control analysis and when comparing all CFS cases to all controls. Several of the diffrentially expressed genes are associated with immunologic functions (e.g., CMRF35 antigen, IL-8, HD protein) and implicate immune dysfunction in the pathophysiology of CFS. These results successfully demonstrate the utility of the blood for gene expression profiling to distinguish subjects with CFS from healthy controls and for identifying genes that could serve as CFS biomarkers.

Objectives. Polymorphisms in human papillomavirus (HPV) type 16 have been shown to be related to geographic areas and are broadly classified as European (E), African (Af), Asian (As), or Asian-American (AA). Certain variants have been reported as being more likely to cause cervical disease; our objectives were to identify new HPV16 polymorphisms, to determine the linkage of the E2 and E6/E7 regions and to determine the minimum sequence necessary to classify variants.

Acute infection is known to perturb psycho-neuroendocrine-immune (PNI) gene expression. Oligonucleotide microarrays were used to examine PNI gene expression in the peripheral blood of 13 subjects with infectious mononucleosis (IM). Novel peripheral blood gene expression activity was correlated with central-nervous-system-mediated symptoms including fatigue and sleep disturbance. Of note, expression of the MADS box transcription enhancer factor 2 polypeptide C (MEF2C) gene, previously implicated in skeletal muscle myogenesis, correlated with symptoms of musculo-skeletal pain and fatigue. Expression of the hypocretin/orexin receptor HCRTR2, which has been implicated in narcolepsy, correlated with sleep disturbance. And, VACHT, the vesicular acetylcholine transporter, was highly correlated with neurocognitive disturbance. The expression of both HCRTR2 and MEF2C in the peripheral blood was validated by reverse transcription PCR. Thus, investigation of the PNI response in peripheral blood may provide novel insights into the complex pathophysiology of centrally mediated disease states.

Before gene expression profiling with microarray technology can be transferred to the diagnostic setting, we must have alternative approaches for synthesizing probe from limited RNA samples, and we must understand the limits of reproducibility in interpreting gene expression results. The current gold standard of probes for use with both microarrays and high-density filter arrays are synthesized from 1 g of purified poly(A)؉ RNA. We evaluated two approaches for synthesizing cDNA probes from total RNA with subsequent hybridization to high-density filter arrays: 1) reverse transcription (RT) of 5 g total RNA and 2) RT-polymerase chain reaction (RT-PCR) of 1 g total RNA, using the SMART system. The reproducibility of these two approaches was compared to the current gold standard. All three methods were highly reproducible. Triplicate experiments resulted in the following concordance correlation coefficients to evaluate reproducibility: 0.88 for the gold standard, 0.86 for cDNA probe synthesized by RT from total RNA, and 0.96 for the SMART cDNA probe synthesized from total RNA. We also compared the expression profile of 588 genes for the total RNA methods to that obtained with the gold standard. Of 150 positive genes detected by the gold standard, 97 (65%) were detected by cDNA probe synthesized by RT of total RNA, and 122 (81%) were detected by the SMART cDNA probe. We conclude that SMART cDNA probe produces highly reproducible results and yields gene expression profiles that represent the majority of transcripts detected with the gold standard.

We have developed a procedure to amplify mRNA into sense RNA (sRNA) so as to create a regenerating biorepository representing the complex mRNA profile in the original sample. The procedure exploits the template-switching activity of reverse transcriptase to incorporate RNA polymerase binding sites upstream of single-stranded cDNA (ss cDNA). Limited PCR was used for double-stranded DNA (dsDNA) synthesis. sRNA was synthesized from PCR products by in vitro transcription (IVT). sRNA was evaluated by real-time reverse transcription (RT)-PCR. sRNA synthesis was successful with RNA from human cell lines and tissues, yielding 2000-to 2500-fold amplification of glyceraldeyde-3 phosphate dehydrogenase (G3PDH). The size of sRNA ranged from 3.0 to 0.1 kb. sRNA synthesis preserved the relative differences in plant mRNAs spiked at abundance ranging over 5 orders of magnitude (0.00001-0.1%). This reflects the high fidelity of sRNA synthesis for mRNA as low as 0.3 copies/cell. sRNA is amplified synthetic mRNA in the 5Ј33Ј direction; the appropriate template for any gene expression analysis.

Molecular events occurring with high-risk human papillomavirus (HPV)-associated dysplastic differentiation of cervical epithelial cells are largely unknown. This study used differential display PCR to identify expression changes between nondifferentiating monolayer and differentiated organotypic (raft) cultures of W12 keratinocytes. These cells were originally derived from a clinical biopsy of HPV 16-positive dysplastic cervical epithelium and retain high-risk HPV 16 and the ability to differentiate, albeit with dysplastic morphology. Using this model system we identified 84 genes with changed expression during dysplastic differentiation. Most (70/84, c80%) were down-regulated with differentiation, consistent with a restriction of expression during terminal differentiation. Twenty-two genes had no known function and 6 novel expressed sequence tags were identified among this group. Of the 62 genes with known functions, 25 belonged to transcription-, translation-, and posttranslation-related categories and 30 had functions associated with neoplastic initiation/progression, calcium signaling, epithelial differentiation, and structure remodeling. Some of the genes with altered expression identified in this model of dysplastic differentiation may be useful biomarkers for early detection of cervical neoplasia and other HPV-associated oropharyngeal and anogenital cancers. Published by Elsevier Inc.

Chronic fatigue syndrome (CFS) is a debilitating disorder of unknown etiology with no known lesions, diagnostic markers or therapeutic intervention. The pathophysiology of CFS remains elusive, although abnormalities in the central nervous system (CNS) have been implicated, particularly hyperactivity of the serotonergic (5-hydroxytryptamine; 5-HT) system and hypoactivity of the hypothalamic-pituitary-adrenal (HPA) axis. Since alterations in 5-HT signaling can lead to physiologic and behavioral changes, a genetic evaluation of the 5-HT system was undertaken to identify serotonergic markers associated with CFS and potential mechanisms for CNS abnormality. A total of 77 polymorphisms in genes related to serotonin synthesis (TPH2), signaling (HTR1A, HTR1E, HTR2A, HTR2B, HTR2C, HTR3A, HTR3B, HTR4, HTR5A, HTR6, and HTR7), transport (SLC6A4), and catabolism (MAOA) were examined in 137 clinically evaluated subjects (40 CFS, 55 with insufficient fatigue, and 42 non-fatigued, NF, controls) derived from a population-based CFS surveillance study in Wichita, Kansas. Of the polymorphisms examined, three markers (À1438G/A, C102T, and rs1923884) all located in the 5-HT receptor subtype HTR2A were associated with CFS when compared to NF controls. Additionally, consistent associations were observed between HTR2A variants and quantitative measures of disability and fatigue in all subjects. The most compelling of these associations was with the A allele of À1438G/A (rs6311) which is suggested to have increased promoter activity in functional studies. Further, in silico analysis revealed that the À1438 A allele creates a consensus binding site for Th1/E47, a transcription factor implicated in the development of the nervous system. Electrophoretic mobility shift assay supports allele-specific binding of E47

Polymorphisms in human papillomavirus type 16 (HPV16) result in variants from the prototype sequence which can be designated according to geographic distribution and are broadly classified as European (E), African (Af), Asian (As), or Asian-American (AA). Detection of variants has been used to distinguish persistent HPV16 infection from re-infection in natural history studies, and variants have been associated with an increased risk of cervical disease in some populations. Variant determination usually relies on conventional Sanger sequencing of regions of the viral genome, with the major variant group assignments requiring the sequencing of only seven polymorphic sites spread over a 242-bp region of the E6 gene. We applied pyrosequencing to facilitate rapid sequencing and enable the simultaneous detection of multiple variants. A single-stranded template for pyrosequencing was prepared by amplifying a 314-bp fragment (nt 75-388) with a biotin at the 5 -end of the reverse primer to facilitate strand separation and purification. Polymorphisms at the nucleotide sites 109, 131, 132, 143, 145, 178 and 350 were determined in three separate sequencing reactions, one of which was a multiplex format. Pyrosequencing of 97 HPV16-positive exfoliated cervical samples confirmed the Sanger sequencing results; however pyrosequencing identified additional variants in several samples containing mixed variants. Published by Elsevier B.V.

While infection with high-risk HPV is the most important risk factor for cervical cancer, HPV alone is insufficient. Our purpose was to identify viral and epidemiologic factors associated with cervical disease in HPV-16 DNA-positive women referred to colposcopy. We used a standardized interview to collect epidemiologic data from consenting women. Total nucleic acids from exfoliated cervical cells were used for all viral assays (HPV detection and typing using L1 consensus PCR with line probe hybridization, variant classification by sequencing, viral load and transcript copy determination by quantitative PCR and transcript pattern by nested RT-PCR). Cervical disease was based on colposcopic biopsy. Logistic regression was used to calculate ORs with 95% CIs. There were 115 HPV-16 positive women among 839 enrollees. By univariate analyses, age >25 years (OR = 3.05, 95% CI 1.20–7.76), smoking (OR = 3.0, 95% CI 1.19–7.56), high viral load (OR = 5.27, 95% CI 2.05–13.60), detection of both E6 and E6*I transcripts (OR = 10.0, 95% CI 2.1–47.58) and high transcript copies (OR = 5.56, 95% CI 2.05–13.60) were significant risk factors for CIN III with reference to No CIN/CIN I. Less than a third of the women (31.5%) had prototype HPV-16 detected, and variants showed no association with disease, viral load or transcription. Viral DNA and transcript copies were highly correlated, and the ratio of transcript copies to DNA copies was not changed with disease status. While viral load, transcript copies and transcript pattern were statistically associated with CIN III, none of these measures effectively discriminated between HPV-16 women with disease requiring treatment and those who could be followed. Cellular proliferation and differentiation pathways affected by HPV should be investigated as biomarkers for cervical cancer screening. © 2005 Wiley-Liss, Inc.

Human papillomavirus (HPV) is a necessary but insufficient cause of cervical cancer. Factors influencing transcription, such as epigenetic silencing through viral DNA methylation, may impact neoplastic progression. Pyrosequencing technology was applied to quantify methylation at 19 cytosine guanine dinucleotide (CpG) sites in the L1 3 and long control region (LCR) of HPV 16 DNA using cell lines, CaSki (∼400 integrated copies of HPV 16) and SiHa (1-2 integrated copies of HPV 16) that differ in their transcriptional activity. Methylation levels ranged from 20 to 100% in CaSki and from 0 to 85% in SiHa over the entire 19 CpG sites, with a >40-fold difference in the methylation levels of their promoter and enhancer regions (SiHa < 2% and CaSki 79%). The method was successful at a limiting dilution of 1-4 HPV 16 DNA copies/3000 cells, a level compatible with most clinical samples. The results were not affected by fixation in methanol-based liquid cytology collection fluid or method of extraction. Conditions optimized with cell lines were applicable to fixed exfoliated cervical cells. Pyrosequencing provides a quantitative site-specific assessment of methylation at multiple CpG sites without cloning, and is thus suited to large-scale molecular epidemiologic studies. Published by Elsevier B.V.

We used differential-display PCR of peripheral blood mononuclear cells (PBMCs) to search for candidate biomarkers for chronic fatigue syndrome (CFS). PBMCs were collected from a subject with CFS and an age- and sex-matched control before and 24 h after exercise. RNA expression profiles were generated using 46 primer combinations, and the similarity between the individuals was striking. Differentially expressed bands were excised, reamplified, and sequenced, yielding 95 nonredundant sequences, of which 50 matched to known gene transcripts, 38 matched to genes with unknown functions, and 7 had no similarity to any database entry. Most (86%) of the differences between the two subjects were present at baseline. Differential expression of ten genes was verified by real-time reverse-transcription PCR: five (cystatin F, MHC class II, platelet factor 4, fetal brain expressed sequence tag, and perforin) were downregulated, and the remaining five genes (cathepsin B, DNA polymerase ε4, novel EST PBMC191MSt, heparanase precursor, and ORF2/L1 element) were upregulated in the subject with CFS. Many of these genes have known functions in defense and immunity, thus supporting prior suggestions of immune dysregulation in the pathogenesis of CFS. Differential-display PCR is a powerful tool for identification of candidate biomarkers. Investigation of these markers in samples from well-designed epidemiological studies of CFS will be required to determine the validity of these candidate biomarkers. The real-time reverse-transcription PCR assays that we developed for assay of these biomarkers will facilitate high-throughput testing of these additional samples.

A gene (inrpk1) encoding a putative receptor-like protein kinase was isolated from the Japanese morning glory, Ipomoea (Pharbitis) nil Roth. cv. Violet. The receptor-like portion of the largest derived polypeptide contains 26 direct leucine-rich repeats (LRRs) in a single block, and the catalytic portion has all the conserved amino acid residues characteristic of Ser/Thr protein kinases. RNA blot analysis detected multiple transcripts in cotyledons. The largest (4.4 kb) transcript encodes the predicted full length polypeptide (INRPK1), whereas a 1.6 kb transcript apparently originates from a secondary transcription initiation site within the gene and potentially encodes a protein kinase identical to INRPK1, but lacking most of the LRRs. Two transcripts (ca. 2.7 and 2.6 kb) are created by alternative 3′-splicing of a large (ca. 1.4–1.5 kb) cryptic intron in the LRR region, creating one transcript (2.6 kb) potentially encoding a small, secretable polypeptide. The larger transcript encoding a polypeptide identical to INRPK1, but lacking 21 LRRs, predominates in vegetative roots. Competitive PCR indicates that inrpk1 mRNA increases 20-fold in cotyledons in response to a previously given single floral-inducing short-day (SD). No differences of this magnitude were detected in any other organs examined from plants similarly treated. This pattern of expression and differential processing suggests a role for inrpk1 in some aspect of SD photoperiodic-induced flowering in morning glory.