A successful implementation of extensive multiplex PCR amplification in DGGE-based gene mutational scanning tests requires a high-resolution separation system. An example of such a system is the original two-dimensional DGGE format31 in which fragments are first separated according to size and, subsequently, further sorted as a function of their melting temperature. Although high resolution may also be obtained by using one-dimensional denaturing gradient gels, the 2-D system allows characterization of each fragment on the basis of its xy-coordinates (size and melting temperature). In practice, by using the 2-D system, it is possible to completely visualize all fragments corresponding to an entire gene or multiple genes for a particular DNA sample and immediately recognize each exon and variants therein (schematically depicted for the BRCA1 gene in FIG. 2). Different sequence

 

FIGURE 2. Empirical two-dimensional gene scanning pattern of the gene BRCA1. All coding exons were amplified from genomic DNA obtained from blood of a normal individ- ual. The sample was found to contain polymorphisms in exons 3 and 20. (Modified after van Orsouw et al.29)

variants in the same exon can be distinguished on the basis of their unique 4-spot pat- terns.29 A potential hindrance to the widespread application of this system to multi- ple, novel genes involves the difficulties in the design of PCR primers generating single-domain fragments which can be resolved under one set of electrophoretic conditions. In order to minimize the time needed to optimize TDGS tests with re- spect to PCR conditions, melting behavior and distribution of fragments in the 2-D gel, a computerized test design has been developed.37

An automated system for 2-D DNA electrophoresis has been This sys- tem has a modest throughput of only 4 2-D gels per day. However, with one technician who can easily operate up to three of these systems a total of 140,000 bp of genomic DNA can be comprehensively analyzed per day for all possible sequence variants. The system is presently modified to increase the throughput per instrument to about 500,000 bp per day.

To test the general applicability and usefulness of TDGS in generating complete spectra of sequence variation in large genes or in multiple genes, we screened cen- tenarian and control populations, on a pilot scale, for functional variants of BRCA1.

TABLE 2. Polymorphisms defining haplotypesa

Exon 11 Exon16 Type 1186 2196 2731 3232 3238 3667 4956 a A G C A G A A b A G T G G G G c A A T G G G G d A G T A G A A e A G C A A A A f G G C A G A A

aOut of 256 possible haplotypes.

COMMON BRCA1 GENE VARIANTS AND SUSCEPTIBILITY TO BREAST CANCER IN CENTENARIANS

BRCA1 is the first human familial breast cancer susceptibility gene As mentioned above and summarized in TABLE 1, BRCA1 is a tumor suppressor gene likely to be involved in DNA recombinational repair.39 A large number of distinct heritable mutations in BRCA1 have been described, most of which result in a trun- cated BRCA1 protein.40 Heritable mutations in BRCA1 confer an over 90% life time risk of breast cancer. In view of the large size of the gene (24 exons, comprising 5592 bp of coding DNA) large-scale gene sequence comparisons in population-based studies have not been performed. However, the data available suggest that the fre- quency of highly penetrant BRCA1 mutations in the normal population is between 1 in 500 and 1 in 2000 individuals.41 The lack of cost-efficient re-sequencing technol- ogy has also constrained population studies on common polymorphisms in BRCA1, which could confer more modest individual risk. In principle, such common variants could explain a far larger part of breast cancer cases.

As mentioned above, one of the benefits of parallel screening by TDGS is that each pattern reveals all homo- and heterozygous variants in the entire gene. The ef- fect of association studies on functional gene variants will now be much more valu- able than the study of single polymorphisms in candidate genes, as has been done so far. So far, a total of 7 polymorphic sites in 6 TDGS fragments in BRCA1 have been identified that give rise to an amino acid change. Owing to strong linkage disequi- librium, these 7 polymorphic sites generate only 6 different haplotypes, i.e., alleles (A–F; TABLE 2). The frequencies of these alleles were compared between groups of centenarians and controls. Centenarians were selected for this study because breast cancer is extremely rare in such individuals of highly advanced age. Hence, it is pos- sible that optimal sequence variants of this gene contribute to this extreme resistan- cy, possibly as a consequence of its increased proficiency in the process of recombinational repair. Two different centenarian populations were studied, i.e., one population consisted of 102 centenarians from the New England area42 and one pop- ulation consisted of 84 centenarians from Italy.43 The control population consisted of 97 individuals from Italy. All three populations were mainly white.

Frequency (%)706050402010        (HH Centenarians It (n=168)[Hl Controls tt (n=194)[17 Breasvovarian cancer probands(Shattuck-Eidens et al, 1997) (n=1590)                   arians NE (n=198)1994) (n=650)= (n=54)1994) (n=322)     I   Haplotype/alleleAPOE4 

FIGURE 3. BRCA1 allele frequencies in two different centenarian populations and one control population (NE = New England; IT = Italy). The allele frequency of APOE4 has been determined in the same populations as a control and was compared with the results of Schächter et al.22