A low-cost, high-throughput, automated single nucleotide polymorphism assay for forensic human DNA applications.

Single nucleotide polymorphism (SNP) analysis of human DNA for the purpose of identification has some promising attributes. The question of approach is critical to the eventual adoption of this technology. The use of a low-volume open array platform was tested with a small selected set of eight SNP primers that have a low F(ST) (the proportion of the total genetic variance contained in a subpopulation [S subscript] relative to the total genetic variance [T subscript]) in human populations. Because multiple SNPs must be interrogated, issues concerning DNA concentration, total DNA, and whole genome amplification were investigated. Excellent correlations were obtained for seven of the eight SNP assays on a set of DNA samples of known configuration over a broad concentration range spanning 25-150ng/microl in blind studies. These seven SNP assays were then applied to 39 DNA samples in a population from southern India. These SNPs were sufficient to individualize each member of this sample population. In a paternity study, these same SNPs showed clear parental relationships. For low amounts of genomic DNA, the use of a commercially available whole genome amplification kit showed promise for genotyping sub-nanogram samples. Discrimination against nonhuman DNA was also demonstrated successfully. Because of the very low quantities of reagents used in the assay, the cost per test becomes reasonably inexpensive. Overall, using commercially available SNP assays, the OpenArray platform showed excellent promise as a highly automated, low-volume, high-throughput system for SNP analysis with potential applications to relevant forensic analyses such as identification and paternity.

A noninvasive approach for assessing tumor hypoxia in xenografts: developing a urinary marker for hypoxia.

Tumor hypoxia modifies the efficacy of conventional anticancer therapy and promotes malignant tumor progression. Human chorionic gonadotropin (hCG) is a glycoprotein secreted during pregnancy that has been used to monitor tumor burden in xenografts engineered to express this marker. We adapted this approach to use urinary beta-hCG as a secreted reporter protein for tumor hypoxia. We used a hypoxia-inducible promoter containing five tandem repeats of the hypoxia-response element (HRE) ligated upstream of the beta-hCG gene. This construct was stably integrated into two different cancer cell lines, FaDu, a human head and neck squamous cell carcinoma, and RKO, a human colorectal cancer cell line. In vitro studies showed that tumor cells stably transfected with this plasmid construct secrete beta-hCG in response to hypoxia or hypoxia-inducible factor 1alpha (HIF-1alpha) stabilizing agents. The hypoxia responsiveness of this construct can be blocked by treatment with agents that affect the HIF-1alpha pathways, including topotecan, 1-benzyl-3-(5'-hydroxymethyl-2'-furyl)indazole (YC-1), and flavopiridol. Immunofluorescent analysis of tumor sections and quantitative assessment with flow cytometry indicate colocalization between beta-hCG and 2-(2-nitro-1H-imidazol-1-yl)-N-(2,2,3,3,3-pentafluoropropyl)acetamide (EF5) and beta-hCG and pimonidazole, two extrinsic markers for tumor hypoxia. Secretion of beta-hCG from xenografts that contain these stable constructs is directly responsive to changes in tumor oxygenation, including exposure of the animals to 10% O2 and tumor bed irradiation. Similarly, urinary beta-hCG levels decline after treatment with flavopiridol, an inhibitor of HIF-1 transactivation. This effect was observed only in tumor cells expressing a HRE-regulated reporter gene and not in tumor cells expressing a cytomegalovirus-regulated reporter gene. The 5HRE beta-hCG reporter system described here enables serial, noninvasive monitoring of tumor hypoxia in a mouse model by measuring a urinary reporter protein.

Use of TP53 reference materials to validate mutations in clinical tissue specimens by single-strand conformational polymorphism analysis.

BACKGROUND: As genetic information moves from basic research laboratories in to the clinical testing environment, there is a critical need for reliable reference materials for the quality assurance of genetic tests. A panel of 12 plasmid clones containing wild-type or point mutations within exons 5-9 have been developed as reference materials for the detection of TP53 mutations. AIM: The goal of this study was to validate the reference materials in providing quality assurance for the detection of TP53 mutations in clinical specimens. METHODS: We studied 33 gynecological samples, 11 apparently normal samples and 22 malignant tumors of various origins. Mutations were identified using single-strand conformational polymorphism analysis with both slab gel and capillary electrophoresis. All DNA samples were amplified with fluorescently labeled PCR primers specific for exons 5-9 for mutation detection. RESULTS: Of the 33 patient samples tested, mutations and polymorphisms were found in six specimens in three of the five exons scanned; no mutations were found in exons 7 or 9. Both a mutation and polymorphism were found in non-malignant specimens from the control group. The mutations were confirmed by DNA sequence analysis of the regions scanned. CONCLUSIONS: Mutations and polymorphisms were detected in the clinical samples. All of the mutations were silent except for one non-conservative mutation in exon 5, codon 181. This study demonstrates the usefulness of the National Institute of Standards and Technology (NIST) TP53 reference panel in TP53 mutation detection in clinical tissue specimens.

Social support and symptoms of postpartum depression among new mothers in Eastern Turkey.

AIM: The aim of the present study was to examine the relationship between symptoms of postpartum depression and social support in new mothers in a semi-rural province (Malatya) of Eastern Turkey. METHODS: This is a descriptive, cross-sectional study. The study was conducted with a 12-item Multidimensional Scale of Perceived Social Support (MSPSS) questionnaire, a 10-item Edinburgh Postnatal Depression Scale (EPDS) questionnaire, and a 16-item demographic/obstetric questionnaire designed by the authors. 364 women who were between 6 to 48 weeks postpartum were included in the study. RESULTS: Symptoms of postpartum depression were negatively correlated with social support (-0.39, P = 0.000). The frequency of the prevalence of symptoms of postpartum depression was 33.2%. The study showed that EPDS mean score was related to several factors, including age, woman's education, woman's occupation, socioeconomic status of family, spouse's education, number of years married, parity, planned pregnancy, method of delivery, knowledge of infant care, sharing of problems with a close person, past psychiatric history and family support during the postnatal period in an Eastern province of Turkey. CONCLUSION: Symptoms of postpartum depression were negatively correlated among Turkish women living in the Malatya province of Eastern Turkey and were associated with the level of social support. The prevalence of postpartum depression was higher than in the published reports regarding most regions of Turkey, with the exception of Northeastern Turkey.

Women's douching practices and related attitudes in eastern Turkey.

AIM: Vaginal douching is a common hygiene practice for many women all over the world, but it is associated with several health risks. Little is known about the beliefs and attitudes that promote and maintain douching practices. Therefore, the objective of the present study was to evaluate the status of vaginal douching practices of women in the Malatya province of eastern Turkey. METHODS: This was a cross-sectional study. A sample of 465 Muslim women was interviewed to ascertain the status of vaginal douching practices. All participants were between 15 and 49 years of age and all were married. Data were collected by using a questionnaire in the process of conducting face-to-face interviews in June 2004. Data analysis included descriptive statistics and logistic regression modeling. RESULTS: The present study revealed that the frequency of douching was 61.5% among women. The participants were frequently douching for feminine hygiene (47.6%). Vaginal douching practices were associated with several factors including education level (odds ratio [OR] = 1.991, 95% confidence interval [CI] = 1.154-3.434), family income (OR = 0.996, 95% CI = 0.994-0.998), marital age (OR = 0.402, 95% CI = 0.223-0.715), frequency of sexual intercourse (OR = 2.335, 95% CI = 1.532-3.554), and presence of genital syndromes/infections (OR = 1.813, 95% CI = 1.215-2.739). CONCLUSION: This study provides preliminary information about women's douching practices and attitudes in the Malatya region of Turkey. It may also provide information to health-care practitioners in their efforts to educate women on the adverse effects of vaginal douching.