Predominance of null mutations in ataxia-telangiectasia

Ataxia-telangiectasia (A-T) is an autosomal recessive disorder involving cerebellar degeneration, immunodeficiency, chromosomal instability, radiosensitivity and cancer predisposition. The responsible gene, ATM, was recently identified by positional cloning and found to encode a putative 350 kDa protein with a Pl 3-kinase-like domain, presumably involved in mediating cell cycle arrest in response to radiation-induced DNA damage. The nature and location of A-T mutations should provide insight into the function of the ATM protein and the molecular basis of this pleiotropic disease. Of 44 A-T mutations identified by us to date, 39 (89%) are expected to inactivate the ATM protein by truncating it, by abolishing correct initiation or termination of translation, or by deleting large segments. Additional mutations are four smaller in-frame deletions and insertions, and one substitution of a highly conserved amino acid at the Pl 3-kinase domain. The emerging profile of mutations causing A-T is thus dominated by those expected to completely inactivate the ATM protein. ATM mutations with milder effects may result in phenotypes related, but not identical, to A-T.      

High throughput parallel analysis of hundreds of patient samples for more than 100 mutations in multiple disease genes

As more mutations are identified in genes of known sequence, there is a crucial need in the areas of medical genetics and genome analysis for rapid, accurate and cost-effective methods of mutation detection. We have developed a multiplex allele-specific diagnostic assay (MASDA) for analysis of large numbers of samples (> 500) simultaneously for a large number of known mutations (> 100) in a single assay. MASDA utilizes oligonucleotide hybridization to interrogate DNA sequences. Multiplex DNA samples are immobilized on a solid support and a single hybridization is performed with a pool of allele-specific oligonucleotide (ASO) probes. Any probes complementary to specific mutations present in a given sample are in effect affinity purified from the pool by the target DNA. Sequence-specific band patterns (fingerprints), generated by chemical or enzymatic sequencing of the bound ASO(s), easily identify the specific mutation(s). Using this design, in a single diagnostic assay, we tested samples for 66 cystic fibrosis (CF) mutations, 14 beta-thalassemia mutations, two sickle cell anemia (SCA) mutations, three Tay-Sachs mutations, eight Gaucher mutations, four mutations in Canavan disease, four mutations in Fanconi anemia, and five mutations in BRCA1. Each mutation was correctly identified. Finally, in a blinded study of 106 of these mutations in > 500 patients, all mutations were properly identified. There were no false positives or false negatives. The MASDA assay is capable of detecting point mutations as well as small insertion or deletion mutations. This technology is amenable to automation and is suitable for immediate utilization for high-throughput genetic diagnostics in clinical and research laboratories.      

Preliminary observations on polar body extrusion and pronuclear formation in human oocytes using time-lapse video cinematography

In this study, we have used time-lapse video cinematography to study fertilization in 50 human oocytes that had undergone intracytoplasmic sperm injection (ICSI). Time-lapse recording commenced shortly after ICSI and proceeded for 17-20 h. Oocytes were cultured in an environmental chamber which was maintained under standard culture conditions. Overall, 38 oocytes (76%) were fertilized normally, and the fertilization rate and embryo quality were not significantly different from 487 sibling oocytes cultured in a conventional incubator. Normal fertilization followed a defined course of events, although the timing of these events varied markedly between oocytes. In 35 of the 38 fertilized oocytes (92%), there were circular waves of granulation within the ooplasm which had a periodicity of 20-53 min. The sperm head decondensed during this granulation phase. The second polar body was then extruded, and this was followed by the central formation of the male pronucleus. The female pronucleus formed in the cytoplasm adjacent to the second polar body at the same time as, or slightly after, the male pronucleus, and was subsequently drawn towards the male pronucleus until the two abutted. Both pronuclei then increased in size, the nucleoli moved around within the pronuclei and some nucleoli coalesced. During pronuclear growth, the organelles contracted from the cortex towards the centre of the oocyte, leaving a clear cortical zone. The oocyte decreased in diameter from 112 to 106 microm (P < 0.0001) during the course of the observation period. The female pronucleus was significantly smaller in diameter than the male pronucleus (24.1 and 22.4 microm respectively, P = 0.008) and contained fewer nucleoli (4.2 and 7.0 respectively, P < 0.0001). After time-lapse recording, oocytes were cultured for 48 h prior to embryo transfer or cryopreservation. Embryo quality was related to fertilization events and periodicity of the cytoplasmic wave, and it was found that good quality embryos arose from oocytes that had more uniform timing from injection to pronuclear abuttal and tended to have a longer cytoplasmic wave. In conclusion, we have shown that time-lapse video cinematography is an excellent tool for studying fertilization and early embryo development, and have demonstrated that human fertilization comprises numerous complex dynamic events.      

Direct mapping of MHC class II epitopes

Despite technical improvements, the mapping of MHC class II epitopes within complex antigens by genetic or biochemical methods is still laborious and expensive. Here, we describe a simple and fast procedure to directly map T helper cell epitopes within known antigens by bacterial expression cloning. Short antigenic fragments, created by digestion of the coding sequence of the antigen with frequently cutting restriction enzymes, are randomly ligated to the coding sequence of GFP in a bacterial expression vector. Bacteria expressing antigen-GFP fusion proteins are then fed directly to MHC II+ antigen-presenting cells and probed with antigen-specific T cells. Bacterial colonies recognized by T cells are expanded, and the antigenic fragments identified by plasmid extraction and sequence analysis. This direct epitope identification (DEPI) approach offers several advantages. First, bacterial colonies expressing the antigen in frame with GFP are easily detectable by virtue of their green appearance and thereby reduce the screening effort significantly. Second, short antigenic peptides normally unstable in bacteria are highly expressed when fused to GFP. Third, the uniformly high level of expression of short antigenic peptides fused to GFP permits the identification of epitopes even within proteins which are difficult to express in bacteria. Furthermore, by fusing double-stranded oligonucleotides to the GFP gene, crucial amino acids within T cell epitopes may be defined. Thus, this method not only facilitates the identification of T cell epitopes, but also makes it possible to assess the role of individual amino acids for MHC binding or T cell recognition.     

Transitional cell carcinoma of the renal pelvis and ureter: Outcome and patterns of relapse in patients treated with postoperative radiation

We assessed the records of 101 patients with locally advanced transitional cell carcinoma (TCC) of the renal pelvis and ureter treated with postoperative radiation therapy to determine outcome and patterns of failure. Locally advanced disease (i.e., T3–4N0 or N+ disease) was identified in 65 patients. Postoperative radiation was used to treat 86 patients, with a median dose of 35 Gy in 20 fractions over 4 weeks to the tumor bed and regional lymph nodes. There were 15 patients with no residual disease who were offered no further therapy. No patient received postoperative chemotherapy. Prognostic factors were examined using univariate and multivariate analysis, and the patterns of failure were identified after postoperative irradiation. Median follow-up was 9.3 years, during which 76 deaths occurred. The 5-year overall survival was 43% and 10-year survival was 23%. A multivariate analysis identified T3 category, lymph node involvement, and age at diagnosis as significant prognostic factors for survival. Tumor grade was a significant prognostic factor on univariate analysis but not on multivariate analysis. Failure analysis showed that only 36% of patients with locally advanced disease remained relapse free. For this group of patients, distant metastases developed in 53%, and locoregional failured occurred in 35% despite postoperative irradiation. Locoregional failure occurred in 95% of patients with nodal involvement who received postoperative radiation, and 77% of those developed distant relapse. This leads us to conclude that patients with resected locally advanced (T3, T4N0, N+) TCC of the upper urinary tracts have a high risk of relapse and death from disease despite postoperative radiotherapy. Because the main feature of the disease is early distant failure, post-operative chemotherapy is required to improve the outcome for this group of patients.     

The role of pancreatic venous sampling in the localization of occult insulinomas

The palpation and enucleation of occult insulinomas (less than 15 mm) can be a difficult surgical problem even with good arteriographic localization. In the authors' limited experience, confirmation of arteriographic findings by pancreatic venous sampling provided little additional localizing information. However, if arteriography is negative or equivocal, venous sampling can indicate the segment of pancreas to be "blindly" resected if the adenoma is not palpable. Venous sampling may be misleading in polyendocrine syndromes because of the frequency of multiple adenomas and variable hormone production.