Received on February 25, 2004
Accepted on May 17, 2004

Molecular Diagnostics and Genetics

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

¹ Unit of Genomics for Diagnosis of Human Pathologies, University of Milan, H. San Paolo, Milan, Italy
² Unit of Separative Techniques, University of Milan, H. San Paolo, Milan, Italy
³ Laboratory Analytical and Medical Chemistry, Department of Medicine, Surgery and Dental Sciences, University of Milan, H. San Paolo, Milan, Italy
⁴ Department of Ophthalmology and Visual Sciences, University Hospital San Raffaele, Milan, Italy
⁵ Departments of Ophthalmology and Pathology, Columbia University, New York, NY
⁶ Unit of Genomics for Diagnosis of Human Pathologies, University of Milan, H. San Paolo, Milan, Italy and Diagnostica e Ricerca San Raffaele SpA, Milan, Italy

^* To whom correspondence should be addressed. E-mail: cremonesi.laura{at}hsr.it.

Background: Mutations in the retina-specific ABC 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 DHPLC profiling. Subsequently, samples from a cohort of 30 patients affected by various forms of macular degeneration were subjected to DHPLC scanning under the same conditions.

Results: This retrospective validation study not only identified all 132 sequence alterations previously detected by double-gradient denaturing gradient gel electrophoresis but also identified 5 sequence alterations that this approach had missed. Moreover, DHPLC scanning of an additional panel of 30 previously untested patients led to the identification of 26 different mutations and 29 polymorphisms, accounting for 203 sequence 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.