Denaturing HPLC Profiling of the ABCA4 Gene for Reliable Detection of Allelic Variations

transporter (ABCA4) gene have been associated with several forms of macular degenerations. Because the high complexity of the molecular genotype makes the scanning of the ABCA4 gene cumbersome, we describes here the first use of denaturing HPLC (DHPLC) to screen for ABCA4 mutations. Methods: Temperature conditions were designed for all 50 exons based on effective separation of 83 samples carrying 86 sequence variations and 19 mutagenized controls. For validation, samples from 23 previously characterized Stargardt patients were subjected to DH-PLC profiling. Subsequently, samples from a cohort of 30 patients affected by various forms of macular degen-eration were subjected to DHPLC scanning under the same conditions. Results: This retrospective validation study not only identified all 132 sequence alterations previously de-tected by double-gradient denaturing gradient gel elec-trophoresis but also identified 5 sequence alterations that this approach had missed. Moreover, DHPLC scan-ning of an additional panel of 30 previously untested patients led to the identification of 26 different muta-tions and 29 polymorphisms, accounting for 203 se-quence variations on 29 of the 30 patients screened. In total, the DHPLC approach allowed us to identify 16 mutations that had never been reported before. Conclusions: These results provide strong support for the use of DHPLC for molecular characterization of the ABCA4 gene