Presently available methods for screening large genetic regions for unknown point mutations are neither flawless nor particularly efficient. We describe an approach, especially well suited to identiyng mutations present in the heterozygous state, that combines several improvements in a protocol called fluorescence-assisted mmath analysis (FAMA). Appropriate gene regions of the wild-type and the putative mutant allele are simultaneously amplified from genomic DNA by using the polymerase chain reaction, and large DNA fragments, so far up to 800 bp, are end labeled with strand-specific fluorophores. Aliquots are denatured and reannealed to form heteroduplexes and subjected to conventional cytosne-and thymine-specific modfifications. Cleavages occuring on opposite strands are detected by denatring gel electrophoresis using an automated DNA sequencer. Since the DNA a nts derived from the mutant allele are also end labeled, the number of informative mispaired bases is doubled compared to conventional searches using wild-type probes. The sensiivty of detection is also increased, because differential fluorescent end labeling allows the identification and measurement ofstand-specific background cleavages at matched cytosine or thymine residues. Automatic superimposition of tracings from different subjects allows mia detection at sites that, because of the nature of the bases involved and of the neighboring sequence, are known to be less susceptible to cleavage. The effects of the latter parameters have been studied quantitatively on a series of point mutations found in the human Cl-inhibitor gene in patents affected by hereditary angioedema. Dilution experiments have demonstrated that most mutations are detected even when the mutant chromosome is diluted 10-fold or more compared with the normal one.The identification of loci responsible for a large number of monogenic diseases and of a growing number of genes involved in tumor predisposition and multifactorial diseases has rapidly expanded the range of application of DNA diagnosis. Since the majority of inherited disorders are heterogeneous at the gene level and are more often due to subtle changes affecting one or several nucleotides than to large deletions or duplications, considerable efforts have been made to develop methods able to detect not only mutations previously identified in probands or known to be prevalent in a given population but also as yet unknown mutations (for reviews, see refs. 1-3). Each of the methods has, however, disadvantages that render its use problematic when searching unknown point mutations within large DNA regions. Denaturing gradient gel electrophoresis (4-6) requires, for example, computer-assisted optimization of the target region and of the electrophoresis conditions, depending on the sequence content of the DNA fragment. The single-strand conformation polymorphism technique (7), in spite of its experimental simplicity, has shown limited sensitivity for the detection of mutations in DNA fragments longer than 200 bp (8, 9).Neith...

Mutations in the Atp7a gene, the mouse homologue of the MNK (ATP7A) gene, have been suggested to be responsible for the mottled phenotype. To date, despite considerable effort, changes associated with the mottled mutations have been detected in only two such mutants. In this study, we identify changes in the level of Atp7a transcript and mutations which could explain the mottled phenotype in nine out of the 10 mutants analysed. The fluorescence-assisted mismatch analysis method used here has proved particularly well suited for mRNA scanning of heterozygous carrier animals, because of its ability to detect mutations even in the presence of an excess of wild-type mRNA. The three new underlying mutations identified at the Atp7a locus include a splice mutation and two missense mutations. While the spectrum of mutations detected in the Atp7a murine gene provides an explanation for at least part of the wide phenotypic variation observed in mottled mutant mice, there is a singular absence of deletions which are associated with a sizeable fraction of human Menkes syndrome cases.

We used the fluorescence-assisted mismatch analysis (FAMA) method to screen rapidly the alpha-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 alpha-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 splicesite 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.