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APOE genotyping: comparison of three methods

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An Erratum to this article was published on 07 April 2009

Abstract

Apolipoprotein E (apo E) polymorphism is associated with increased risk of cardiovascular and Alzheimer diseases, making its genotyping of potentially predictive value. We developed a rapid, reliable and specific method for determining APOE genotypes by fluorescent resonance energy transfer (FRET) over a high number of samples in a single run using a LightTyper™ device and dedicated probes. The method, validated with 75 blood samples, was designed to simultaneously detect three common APOE polymorphisms, ε2, ε3 and ε4, and to identify in a single reaction any of the six following genotypes: ε2/ε2, ε3/ε3, ε4/ε4, ε3/ε4, ε4/ε2, ε3/ε2. The assay involved three phases: (1) DNA extraction, (2) amplification, and (3) melting curve analysis using FRET technique. Briefly, genomic DNA of patients was extracted from total blood. Fragment of APOE was amplified by a first PCR run. Fluorescent labeled probes were added in a second PCR run. FRET genotyping showed following distribution: (1) 1.3% for ε2/ε2 and ε4/ε4 homozygotes, (2) 4.0, 6.6 and 14.7% for ε2/ε4, ε2/ε3 and ε3/ε4 heterozygotes, respectively, and (3) 72.0% for ε3/ε3 homozygotes. Moreover, a careful analysis of the FRET melting curves allowed us to determine the presence of a new polymorphism on the third position of the codon 158 (-AAGCGT-), namely, two nucleotides downstream from the known polymorphism. When the FRET analysis was compared to those obtained by RFLP and sequencing, the presence of this new polymorphism was confirmed only by sequencing thus indicating that RFLP analysis is not always reliable for genotyping.

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Acknowledgments

We would like to thank S. Visvikis-Siest, M. Pfister and Roche Diagnostics (Meylan, France) for technical support.

Conflict of interest statement

The authors declare that they have no conflict of interest related to the publication of this manuscript.

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Authors and Affiliations

  1. EA 3442, Faculté de Pharmacie, 5 rue Albert Lebrun, BP 403, 54001, Nancy Cedex, France

    B. H. Rihn

  2. Laboratoire de Virologie, CHU Nancy-Brabois and Faculté de Pharmacie, Nancy-Université, Nancy, France

    S. Berahmoune & A. Le Faou

  3. Arab European University, Damascus, Syria

    M. Jouma & S. Chamaa

  4. Biological Sciences University Sapienza, Rome, Italy

    L. Marcocci

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  1. B. H. Rihn
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  2. S. Berahmoune
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  3. M. Jouma
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  4. S. Chamaa
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  5. L. Marcocci
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  6. A. Le Faou
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Corresponding author

Correspondence to B. H. Rihn.

Additional information

An erratum to this article can be found at http://dx.doi.org/10.1007/s10238-009-0045-1

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Rihn, B.H., Berahmoune, S., Jouma, M. et al. APOE genotyping: comparison of three methods. Clin Exp Med 9, 61–65 (2009). https://doi.org/10.1007/s10238-008-0012-2

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  • DOI: https://doi.org/10.1007/s10238-008-0012-2

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