Improved HUMARA for the Detection of X-Linked Agammaglobulinemia Carriers.

Background: Fragment analysis of exon 1 of the human androgen receptor, known as HUMARA, is a polymerase chain reaction (PCR)-based method for detecting X-linked agammaglobulinemia (XLA) carriers. This method takes advantage of X-chromosome inactivation (XCI) in female cells. XLA is caused by mutations in the Bruton tyrosine kinase (BTK) gene, located in Xq22.1. In this study, XCI is nonrandom or skewed in B-cells. B-cells with an active X-chromosome carrying a BTK mutation do not mature. Peripheral B-cells in XLA carriers inactivate the mutated X-chromosome. Methods: HUMARA was performed using DNA from purified B-cells and total leukocytes. DNA was digested using methylation-sensitive HhaI. The PCR of the HUMARA polymorphic marker was performed with the HhaI digested samples. The lengths of the PCR products were determined. If a suspected carrier showed skewed XCI in their B-cells, the marker length that corresponded with the length determined in the index patient indicated their carrier status. Results: HUMARA was conducted on purified B-cells; this allowed easier identification of the mutated or inactive allele, as the active allele was enzymatically digested. Analysis of 30 possible carriers using modified HUMARA corroborated that the carrier status in all samples that were heterozygous for the marker using XCI calculation for leukocytes showed a Gaussian distribution, while the carrier B-cell DNA showed a skewed XCI. Conclusion: Carrier status was successfully determined for most of the analyzed samples. B-cell enrichment resulted in precise carrier determination data, reduced the sample size, and facilitated inactive and active allele identification.

Diffusion-weighted magnetic resonance imaging evaluation of intra-abdominal sites of implants to predict likelihood of suboptimal cytoreductive surgery in patients with ovarian carcinoma.

OBJECTIVES: To analyse the diagnostic accuracy and to establish a predictive score based on diffusion-weighted magnetic resonance imaging (DWMRI) compared to exploratory laparotomy (EL) for predicting suboptimal cytoreductive surgery for different intra-abdominal sites of implants in patients with ovarian cancer. METHODS: Thirty-four patients with advanced ovarian carcinoma were studied. Preoperative DWMRI of the abdomen and pelvis was performed. DWMRI findings were compared with EL. Ten anatomical sites were selected for inclusion in the score. Sensitivity, specificity, positive predictive value, negative predictive value and accuracy for suboptimal cytoreduction were calculated for both DWMRI and EL. Receiver operating characteristic (ROC) curve analysis was used to assess the ability to predict suboptimal cytoreduction. RESULTS: Using predictive score, ROC curves were generated with an area under the curve of 0.938 for DWMRI and 0.947 for EL (P < 0.0001). For DWMRI, a score ≥6 had the highest overall accuracy at 91.1 % and identified patients with unnecessary EL with a sensitivity of 75 %. For EL, a score ≥4 had the highest overall accuracy at 88.2 % and was able to identify patients with unnecessary EL with a sensitivity of 87.5 %. CONCLUSIONS: DWMRI is an emerging technique that may be useful to predict suboptimal cytoreduction in ovarian cancer. KEY POINTS: • DWMRI is increasingly used in ovarian cancer. • DWMRI is an accurate technique for depicting intra-abdominal sites of implants • DWMRI is useful for predicting optimal cytoreductive surgical outcome. • We report a high predictive value similar to exploratory laparotomy.

Preneoplastic and neoplastic changes in the Leydig cells population in mice exposed to low doses of cadmium.

The morphological consequences of chronic exposition to low doses of cadmium (Cd) in the Leydig cells population were investigated in 40 sexually mature male mice at morphological and ultrastructural levels. Animals were orally exposed to cadmium (0.015 g/L of CdCl(2) in drinking water) for 3, 6, 12 and 18 months and then sacrificed, samples were collected for toxicological, light and electron microscope studies. Vascular lesions were evident from 6 months of Cd exposure, the severity of the morphological changes observed in the testicular vases were highly and clearly correlated to the time of exposure to Cd. The severity of the Leydig cells morphological changes were increasing along the time of exposure. Presence of cytoplasm vacuolization and degenerative images of the cells were frequent after 12 months of Cd exposure. Also two Leydig cells tumours after 12 and 18 months Cd exposure were presented. These results indicate that prolonged exposures to low doses of Cd are able to induce morphological damage on the Leydig cells.