May 2007
Volume 48, Issue 13
Free
ARVO Annual Meeting Abstract  |   May 2007
Microelectronic Chip Detection of ABCR Gene Mutations in AMD Central Italy Patients
Author Affiliations & Notes
  • C. Battista
    Oculistica, Ospedale Civile S Donato Arezzo, Arezzo, Italy
  • A. Sodi
    Ophtalmology, Eye Clinic, University of Florence, Florence, Italy
  • S. Bernabini
    Genetic Diagnosis, Careggi Universitary Hospital, Florence, Italy
  • I. Passerini
    Genetic Diagnosis, Careggi Universitary Hospital, Florence, Italy
  • A. Mariottini
    Genetic Diagnosis, Careggi Universitary Hospital, Florence, Italy
  • F. Torricelli
    Genetic Diagnosis, Careggi Universitary Hospital, Florence, Italy
  • A. Romani
    Oculistica, Ospedale Civile S Donato Arezzo, Arezzo, Italy
  • U. Menchini
    University of Florence, Eye Clinic, Florence, Italy
  • Footnotes
    Commercial Relationships C. Battista, None; A. Sodi, None; S. Bernabini, None; I. Passerini, None; A. Mariottini, None; F. Torricelli, None; A. Romani, None; U. Menchini, None.
  • Footnotes
    Support None.
Investigative Ophthalmology & Visual Science May 2007, Vol.48, 3006. doi:
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      C. Battista, A. Sodi, S. Bernabini, I. Passerini, A. Mariottini, F. Torricelli, A. Romani, U. Menchini; Microelectronic Chip Detection of ABCR Gene Mutations in AMD Central Italy Patients. Invest. Ophthalmol. Vis. Sci. 2007;48(13):3006.

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      © ARVO (1962-2015); The Authors (2016-present)

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Abstract

Purpose:: ABCR gene encodes the photoreceptor-specific ATP-binding cassette transporter and it is involved in recessive Stargardt macular distrophy, it has been claimed that heterozygotes for ABCR mutations are predisposed to AMD. We developed a microchip-based assay to identify the most common AMD-associated ABCR gene variants ( G1961E, D2177N ) by use of the Nanogen Workstation; subsequently we report the results achieved using this chip in an ethnically homogeneous group of AMD patients.

Methods:: For each mutation we designed a set of probes consisting of a forward and a reverse PCR primer , two reporter oligonucleotides and one stabiliser oligonucleotide. We optimized Nanogen protocols determining the more suitable chemical and thermal stringencies. The chip assay was validated on 65 Stargardt patients previously characterized by standard methods ( DHPLC, automatic sequencing ) with complete concordance of results. We then studied a group of 85 AMD patients coming from Tuscany, a region of Central Italy and our series was ethnically homogeneous For all patients, genomic DNA was isolated from peripheral blood, exons 42 and 48 of ABCR gene were amplified in multiplex PCR; biotinylated amplicon was electronically addressed on the chip to selected test site. The DNA at each test site was then hybridized to a mixture of fluorescently labelled wild-type or mutant probes specific for the first mutation of interest. Quantitative analysis of the hybridation results was performed by use of dedicated software. After the first hybridization and scanning procedure, a stripping step was performed to remove reporters and to allow hybridization with the second set of probes.

Results:: In our series three subjects out of 85 (3,5%) were carriers of one of the considered ABCR mutations (two heterozygotes for G1961E and one heterozygote for D2177N).

Conclusions:: Nanogen technology is a high-throughput metod, suitable for large scale studies. Our AMD microchip is a new quik molecular diagnostic system useful to perform many genetic characterizations in a short time. In our study it was used for studying G1961E and D2177N ABCR mutations in a group of AMD Central Italy patients,detecting one in these mutations in one allele of 3/85 patients (3,5%). The prevalence of these two sequence changes in our population was in agreement with that one reported in previous studies (Allikmets R 2000).

Keywords: gene screening • age-related macular degeneration • clinical (human) or epidemiologic studies: prevalence/incidence 
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