Denaturing high-performance liquid chromatography (DHPLC) as a fast tool to screen a control population for the occurrence of unclassified variants (UV) in the BRCA1 gene

Excerpt

The identification of BRCA1 and BRCA2 mutations has enabled physicians to identify persons at high risk for carcinoma of the breast and ovary in hereditary breast-ovarian cancer (HBOC) families. Tests for known mutations are very sensitive and specific. The interpretation of previously undescribed variants (UV) is complicated because it could be either a cancer-causing mutation or a polymorphism. Until a functional test is available, general population frequency analysis of unclassified variants in the BRCA1 coding region is useful to support the putative role of missense mutations. In contrast to the very cumbersome evaluation of sequence data, the evaluation of results by DHPLC is quite effortless because the investigator has to discriminate only between single and multiple peaks in the elution profiles. The following reported UVs were analysed with the DHPLC technique: A835G; C1605T; G2531C; A2577G; C2596A; C2640T; C2715T; G3238A; G4654T; T5002C; C5029T; G5075A and 5136 del CAC. The analysis was done with DNA from 98 persons (81 females and 17 males) who had no BRCA1 related carcinomas in their family history. The ages for the females range from 58 to 92 years (median 73 years) and for the males from 58 to 87 years (median 70 years). With this strategy we detected G5075A four times, G3238A two times and C2715T one time. Therefore, these BRCA1 alterations can be considered as rare polymorphisms. The other investigated UVs could not be detected in the samples and the nature of the alterations still remains unclear. With the DHPLC technique we also detected two novel alterations G1606A and G1736A. Sequence comparison with dog BRCA1 reveals that G1606A represents a wild-type constitution and G1736A is a silent mutation. Therefore both alterations represent rare polymorphisms. …