Point mutation analysis of archived cytogenetic slide DNA.

Archived Giemsa-stained cytogenetic slide repositories represent valuable DNA resources for medical, scientific, and forensic studies. Sequencing readily identified a Charcot-Marie-Tooth disease point mutation in a 209-bp PCR amplified product. With optimal PCR primers and amplification conditions, our protocol quickly and reliably isolated sufficient DNA for at least 12 independent PCR amplification reactions for forensic and medical applications from single slides up to 5 years old.

Maternal transmission of Helicobacter pylori in the perinatal period.

OBJECTIVES: Studies indicate that Helicobacterpylori (HP) infection is closely related to gastric mucosa lesions and well-differentiated gastric cancer. In Japan, the HP-positive rate in childhood is 5-6%, which is similar to other developed countries, and in regard to the infection route, oral infection is considered important. To our knowledge there have been no reports on mother-to-child transmission and in this study we investigated maternal HP infection status to determine the potential of mother-to-child transmission in the perinatal period. METHODS: After obtaining informed consent from 1,588 pregnant women, mother's blood and cord blood were collected at delivery to measure HP antibody (Helico-G). Gastric contents from the neonates were cultured to isolate H. pylori (Skirrow medium). Vaginal discharge (73 women) and dental plaque scraping swabs (48 women) were collected before delivery, and milk (66 women) was collected after delivery from 212 HP antibody-positive pregnant women to detect H. pylori by PCR. RESULTS: The HP antibody-positive rate for the pregnant women was 29.2%. H. pylori was not detected in the vaginal discharge from HP antibody-positive pregnant women, but dental plaque scraping swabs from 4 women and milk from 4 women was positive. CONCLUSION: We considered that vertical infection during pregnancy or at delivery is unlikely as a route of mother-to-child HP antibody infection. However, horizontal infection through breast-feeding may occur.

Dual blastomere analysis improves reliability of preimplantation trembler mouse diagnosis.

Dual blastomere biopsy and independent blastomere analysis dramatically improved preimplantation diagnostic reliability as confirmed by testing the remaining biopsied eight-cell mouse embryo. The autosomal dominant trembler mouse point mutation was selected as a model for human preimplantation diagnosis because: (1) single cell assay failure is predicted to be the highest when testing autosomal dominant mutations; (2) point mutations represent the most common of all mutation categories and the most demanding mutation to assay reliably; and (3) the trembler mouse point mutation in peripheral myelin protein 22 (Pmp22) is a model of human Charcot-Marie-Tooth type 1A disease. Mathematical models predict our experimental results assuming amplification of 80% of each target allele as well as trembler sperm DNA contamination in 1 of 44 normal biopsied single blastomeres. Single blastomere analysis correctly predicted the genotype in only 84% of embryos that would have been implanted as normal. In contrast, when independent tests of both biopsied blastomeres agreed, test results were confirmed in 20 of 21 (95.2%) of the remaining six-cell biopsied embryos designated as normal. Thus, biopsied six-cell embryo confirmation demonstrated that dual biopsied blastomere analysis improved test reliability remarkably.