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

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

^aAddress correspondence to this author at: Unit of Genomics for Diagnosis of Human Pathologies, University Hospital San Raffaele, Via Olgettina 58, 20132 Milan, Italy. Fax 39-02-26434767; 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 scanning of the ABCA4 gene cumbersome, we describe 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: DHPLC profiling 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.

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