It took 45 scientists at six research centers more than three years to findthe first gene shown to cause early-onset breast cancer.How did they do it, and who did what?Three of the six centers _ a university, a National Institutes of Health(NIH) institute and a commercial biotechnology company _ sharedmost of the highly collaborative work that began in 1990 with a stretchof DNA marked on a chromosome, and ended last Wednesday with theannouncement that BRCA1, the breast-cancer gene, was at last aknown quantity.All 45 investigators are co-authors of a paper to appear in Science forOct. 7, titled, "Isolation of BRCA1, the 17q-linked breast and ovariancancer susceptibility gene." Its principal author is geneticist MarkSkolnick, who wears two hats, one academic, one industrial.He is co-founder and vice-president for research at Myriad GeneticsInc., and professor of medical informatics at the University of Utah,both in Salt Lake City.Molecular biologist P. Andrew Futreal, a postdoctoral fellow in theLaboratory of Molecular Carcinogenesis at NIH's National Institute ofEnvironmental Health Sciences (NIEHS), of Research Triangle Park,N.C., is first author of a back-to-back companion paper, "BRCA1mutations in primary breast cancer and ovarian carcinomas," slated forthat same Science issue. Futreal reports to Roger Wiseman, who headsthe Comparative Carcinogenesis Group in the NIEHS Laboratory ofMolecular Carcinogensis.Cancer biologist Wiseman, who is principal author of that secondScience paper, said that discovery of the breast-cancer gene "is theclosest thing to magic I've been associated with in my entire scientificcareer."BioWorld interviewed both Skolnick and Futreal to discern how theybrought BRCA1 to light:

BioWorld: What was step one?Skolnick: To find a region genetically on the human genome.Mary Claire King, and others, at the University of Michigan, hadlocalized such a region on chromosome 17 of patients with familialbreast cancer. Building on this data, we constructed a physical map ofthe suspect region.BioWorld: Then how did you proceed?Skolnick: The general concept of the procedure is positionalcloning. We first blanketed that region of the genome in overlappingyeast artificial chromosome [YAK] clones. At the same time wecovered the region with P1 clones, to make a contig composed of bothYAKs and P1-vector clones. The P1 allowed us to clone 80 to 90kilobase fragments at a time.BioWorld: How did you then use these?Skolnick: In several ways. For one, Lilly Research Laboratoriesdid shotgun sequencing on them for us, so we could sample somefragments. For another, we looked at the computer-software exon-trapping program, to see if we had a coding sequence or not. And weidentified such sequences by solution hybrid capture.BioWorld: How does that work?Futreal: It's a fairly straightforward method invented some years agoat the University of Texas. We at NIEHS and Myriad modified it forour breast-cancer search. To pull out genes we took the individualclones that represent the contig of genomic DNA that Mark [Skolnick]just described, and cut them up with restriction enzymes into smallpieces. These we labeled with biotin molecules by polymerase chainreaction [PCR] technique. Next we made a primary complementary DNA frommessenger RNA isolated from normal mammary glands. That gave us apool of breast-expressed genes. Our reasoning was that the cancer-causing gene we were hunting would, by definition, have to beexpressed in breast tissue, so that was a logical place to screen. Then we hybridized that mammary-derived cDNA to ourbiotin-tagged genomic fragments. Any cDNA molecules that did nothybridize stayed in solution. They couldn't find a partner, so remainedfree, not linked with biotin. The next trick was to take magnetic beads coated withstreptavidin, which strongly binds biotin, and throw them into that tubeof solution. An external magnet then separated out all of thebiotinylated molecules. We were left with a solution of cDNA that in some fashionrepresented genes encoded by that genomic DNA. Two rounds of PCRcycling enriched those sequences 10,000-fold for the gene of interest.We were pulling all the genes expressing in the breast that map to theregion of interest.Skolnick: At Myriad we further modified Andy's [Futreal]capture technique to be directional. By walking the overlappingmolecular clones toward the 5' end, it helped us find the rest of theBRCA1 gene. That directional solution hybridization is a new idea.BioWorld: What then?Futreal: At this point, we had what looked like 60 to 65 independentgene fragments, and no way to relate them. Originally, BRCA1 cameout in two pieces that looked independent. But when we put these on northern blots, even though onedidn't look anything like the other, both recognized a 7.8-kilobasemessenger rRNA message. We said, `Aha! 7.8kb messages are kind ofrare anyway.' So at that point we started doing contigs around thesesmall pieces, while at the same time Utah was pulling cDNA's fromconventional screens. That's how the BRCA1 signal was built up _ by acombination of methods.Skolnick: Using these approaches, shotgun sequencing,software exon trapping, hybrid capture and conventional cDNA libraryscreening, we found candidate transcripts _ pieces of putativelyexpressed sequences. From these we assembled a composite sequence for acandidate BRCA1 gene. Based on that, we designed the nucleotideprimers to PCR-amplify genomic DNA from members of familiesknown to carry BRCA1 gene mutations. We then compared their DNAsequences with normal, unmutated BRCA1 sequences. This made itpossible to identify the mutations in the eight individuals, each from adifferent cancer-susceptible family, we screened. We found five mutations, sufficient evidence, so we could saythat this must be the right gene. n091994BRCA1

-- David N. Leff Science Editor

(c) 1997 American Health Consultants. All rights reserved.