Fluorescence-assisted mismatch analysis (FAMA) for exhaustive screening of the α-galactosidase A gene and detection of carriers in Fabry disease |
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Authors: | Dominique Germain M Biasotto Mario Tosi Tommaso Meo Axel Kahn Livia Poenaru |
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Institution: | (1) Laboratoire de Génétique, CHU Cochin Port-Royal, Université René Descartes, Paris, France, FR;(2) INSERM U129, ICGM, 24 rue du Faubourg St Jacques, F-75014 Paris, France, FR;(3) Unité d’Immunogénétique, INSERM U276, Institut Pasteur, 25 rue du Docteur Roux, F-75015 Paris, France, FR;(4) Laboratoire de Génétique et Pathologie Métabolique, CHU Cochin Port-Royal, 24 rue du Faubourg Saint Jacques, F-75014 Paris, France Fax: +33-1-44-41-24-46; e-mail: germain@icgm.cochin.inserm.fr, FR |
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Abstract: | We used the fluorescence-assisted mismatch analysis (FAMA) method to screen rapidly the α-galactosidase A gene in patients
with Fabry disease in order to identify unknown mutations and help define genotype-phenotype correlations in this X-linked
lysosomal storage disorder. Chemical cleavage at mismatches on heteroduplex DNA end-labeled with strand-specific fluorescent
dyes, reliably detects sequence changes in DNA fragments of up to 1.5 kb and locates them precisely. Exhaustive scanning of
the α-galactosidase gene was accomplished on four polymerase chain reaction-generated amplicons, covering all seven exons,
the exon-intron boundaries, and 700 bp of 5′-flanking sequence. Mutations were identified in each of the 15 patients studied
from nine unrelated kindreds. Among the seven previously undescribed sequence changes, three are obviously pathogenic because
they lead to premature protein termination. The other four, a splice-site mutation and three missense mutations, were the
only changes found upon complete scanning of the gene and its promoter. In addition, FAMA also detects female heterozygous
carriers more dependably than direct sequencing, and thus provides a valuable diagnostic test. In Fabry disease, this molecular
criterion is especially important for genetic counseling since heterozygotes can be asymptomatic and their enzymatic values
within the normal range.
Received: 9 April 1996 / Revised: 8 July 1996 |
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