Cloning and Characterization of Expanded CAG-Repeat Containing Sequence(s): Identification of Candidate Breast Cancer Predisposition Gene(s)
Abstract
Genetic factors have been shown to influence the risk of developing breast cancer. To date only two breast cancer predisposition genes, BRCA1 and BRCA2, have been identified. A wide spectrum of BRCA mutations has been found in breast cancer families. However, in the majority of breast cancer cases BRCA1 or BRCA2 mutations were not detected, suggesting the presence of other breast cancer predisposition genes Trinucleotide repeats are present widely in human genome. Expansions of some trinucleotide repeats have been recognized to be the cause of several genetic disorders. In a previous study using the Repeat Expanded Detection (RED) method, we detected expanded (CAG) repeat of repeat sizes of over 144 in 2.4% of breast cancer cases in a sample of 212 patients. No expansion of this magnitude has been detected in 196 population controls samples. Since RED does not provide information about the location of expanded repeats, it is necessary to clone and localize them. In this stage of the project we have developed and optimized an *efficient cloning and screening strategy using a combination of dynabead enrichment microsatellite isolation protocol and the RepeatArray system. We also validated the results by sequencing the inserts. We have shown that applying the dynabead enrichment for a second round dramatically increases the percentage of CAG containing clones (around 90%). We have also detected a large GAG/CTG containing fragment of 65 repeats suggesting that our system is able to identify large repeat containing inserts. We have also established RepeatArray technology, which provides the distinction between smell and large repeat containing fragments and provides a high throughput pre-screening tool and eliminates the need for excess sequencing. Currently, we are at the stage of further validation and application of the strategy to breast cancer.
Document Details
- Document Type
- Technical Report
- Publication Date
- Jun 01, 2004
- Accession Number
- ADA427658
Entities
People
- Hilmi Ozcelik
Organizations
- Mount Sinai Hospital, Toronto