Biomedical subjects

David C Swan

Publications and source records attributed to David C Swan.

6 recordsLinked to original sources

PubMed2006

Quantitation of site-specific HPV 16 DNA methylation by pyrosequencing.

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 ( approximately 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.

Cell Line, Tumor↗

PubMed2006

Simultaneous amplification and identification of 25 human papillomavirus types with Templex technology.

The majority of existing human papillomavirus (HPV) genotyping assays are based on multiplex PCR using consensus or degenerate primers. We developed a Templex HPV assay that simultaneously detects and identifies 25 common HPV genotypes in a single-tube reaction using type-specific primers for the HPV-specific E6 and E7 genes. The analytical sensitivities of the Templex assay for HPV type 16 (HPV-16), -18, and -56 were 20, 100, and 20 copies per reaction mixture, respectively. The Templex assay provides semiquantitative information on each type when multiple HPV types coexist in one reaction. We tested 109 clinical cervical specimens previously evaluated with the Digene HC2 high-risk HPV DNA test and found 95.4% concordance between the assay results. The Templex assay provided type-specific results and found multiple types in 29.2% (14 of 48) of high-risk HPV-positive samples. The entire Templex procedure, including DNA extraction, can be completed within 5 hours, providing a rapid and reliable diagnostic tool for HPV detection and typing that is amenable to automation.

Cervix Uteri↗

PubMed2006

Human papillomavirus in amniotic fluid.

BACKGROUND: There is evidence to suggest that human papillomavirus (HPV) can cross the placenta resulting in in-utero transmission. The goal of this study was to determine if HPV can be detected in amniotic fluid from women with intact amniotic membranes. METHODS: Residual amniotic fluid and cultured cell pellets from amniocentesis performed for prenatal diagnosis were used. PGMY09/11 L1 consensus primers and GP5+/GP6+ primers were used in a nested polymerase chain reaction assay for HPV. RESULTS: There were 146 paired samples from 142 women representing 139 singleton pregnancies, 2 twin pregnancies, and 1 triplet pregnancy. The women were 78% Caucasian, 5% African American, 14% Asian, and 2% Hispanic. The average age was 35.2 years with a range of 23-55 years. All samples were beta-globin positive. HPV was not detected in any of the paired samples. CONCLUSION: Given the age range, race, and ethnicity of the study population, one would anticipate some evidence of HPV if it could easily cross the placenta, but there was none.

Journal Article↗

PubMed2006

Human papillomavirus type 16 variant assignment by pyrosequencing.

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.

Base Sequence↗

PubMed2005

Epidemiologic and viral factors associated with cervical neoplasia in HPV-16-positive women.

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.

Adolescent↗

PubMed2005

Human papillomavirus type 16 E2 and E6/E7 variants.

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. METHODS: We sequenced the complete E2, E6, and E7 regions in all HPV16-positive cervical samples identified in a case-control study of pre-invasive cervical disease. RESULTS: In the 100 samples analyzed, only one new polymorphism was identified, a synonymous change, T3205A, in region E2. The frequency distribution of variants in the sample set was 37 European prototypes and 27 E-G350, 16 AA, 5 Af1, 2 Af2, 8 E-C109G, 3 E-G131G, and 2 As. As shown by others, region E7 varied much less than E6 and E2. CONCLUSIONS: In each case, E2 changes were linked to the expected E6/E7 changes, and there was no evidence for recombination. The linkage between E2 and E6/E7 allows variant classification to be based on a short E6 sequence (nt 109-350).

Base Sequence↗