Remember what a long and difficult process it was to track down thefamilial breast cancer susceptibility gene? Researchers knew it waslocated on a well-defined region of the long arm of chromosome 17,so no one thought the final isolation steps would take very long. Butfour years passed before success finally came in the fall of 1994.(See BioWorld Today, Sept. 15, 16, 19, 1994; p. 1.)

While the search for other genes linked to hereditary diseases is notalways so well-publicized, the task is often no less daunting. But thepotential payoff for the sufferers can be great when isolation of amutant gene leads to fast-track clinical development of therapies, ashas been the case for cystic fibrosis.

In what may be a big step forward in identifying the mutations thatcause hereditary diseases, the Feb. 1 issue of Nature Genetics reportsa signal advance in the ability to reveal the sought-after mutations ofthese diseases. In a paper titled, "Detection of mutations by cleavageof DNA heteroduplexes with bacteriophage resolvases," pathologistRobert Mashal and his co-authors at Brigham and Women'sHospital, Boston, describe the use of enzymes called resolvases tocut and characterize genomic DNA that harbors mutant genes.

Bacteriophage resolvases recognize and digest branched DNAstructures during the viral packaging process that forms mature,infective bacterial viruses. Mashal's group harnessed resolvase'sability to digest mismatched bacteriophage DNA, and used it torecognize the mismatches caused by mutant genes in humangenomic DNA. Once the mismatch is recognized, this enzyme cutsthe DNA at the spot where the mutated gene resides. This localizedcleavage occurs because, in heterologous carriers of the hereditarydisease, the mutant gene on one strand of DNA does not paircompletely with the normal gene on the complementary strand.

In practice, the genomic DNA to be analyzed is polymerize chainreaction-amplified and then digested by a resolvase. After thisdigestion, the DNA is separated on gels and the sizes visualized. Thisallows determination of whether the resolvase has identified and cuta mismatched section of DNA.

In the experiments reported in Nature Genetics, the resolvasesspotted three out of three short deletions and 13 of 14 pointmutations, and detected all four classes of possible single nucleotidemismatches. The sizes and sequences of the cleavage fragmentsgenerated corresponded to those predicted from the mutations used.

Current techniques for resolving aberrant genes rely primarily on therelatively small conformational changes in the double-stranded DNAthat contains mutations large enough to allow electrophoreticseparation on gels. However, point mutations often evade thiscriterion. Alternatively, sequencing techniques are used to locatemutated genes unambiguously, but this is a time-consuming process and not suitable for rapid screening of mutations,Mashal's paper suggests.

As a result of the problems inherent in current techniques, MichaelDean, of the National Cancer Institute's Frederick Cancer ResearchCenter, concluded in an accompanying editorial that this newresolvase method of detecting mutations has the potential for beingas easy, inexpensive, and sensitive as present techniques, with theadvantage of being able to analyze larger genomic DNA fragmentsof up to one kilobase.

"The big picture here," Dean told BioWorld Today, "is that this isthe beginning of the use of enzymes to detect mutations, optimizingthe reaction conditions and the enzymes used will be the next step indeveloping this technique."

Resolvases Good For Quick Screenings

In the near future, rapid identification of new mutations andpolymorphisms in DNA segments should be possible usingresolvase-like enzymes. As Mashal told BioWorld Today,"Resolvases should be great for quick, fine-tuned screening once thelocation of a disease-associated gene has been narrowed down andmapped to a region of a chromosome." So, resolvases do appear tohave the potential to significantly shorten the final isolation stepsthat took so long for the breast cancer-associated genes.

In the long term, those enzyme-based assays for detecting hereditarymutations may also be used in clinical labs that screen patients forthe carriers of genetic diseases. The simplicity of the technique andits adaptability for use with non-toxic and non-radioactive materialspresent significant advantages.

`The hope is to find resolvase-like enzymes, Dean suggested,"possibly mutant ones, that recognize mutant mismatches and sit onthe DNA at the mismatched site. Then antibodies could be developedagainst these enzymes and a fast, efficient ELISA-type microtiterplate assay developed, one that would be very suitable for rapidscreening of carriers in clinical labs."

However, as Mashal pointed out , there is still some way to go beforethis hope is realized because "even though the detection error ratewill likely be small using these assays, resulting in a predictivecertainty of 95 percent or greater, this level of certainty may still notbe good enough in a clinical screening test."

From a commercial standpoint, companies that own rights to, or aredeveloping, resolvase-like enzymes are well-positioned to share inthe success of these techniques for detecting mutated genes. Mashalhas offered to make samples of his purified resolvases available onrequest to researchers, but commercial availability of these DNAmismatch-detecting enzymes is critical for future commercialdevelopment.

Applied Technology Genetics Corporation (ATGC), of Malvern, Pa.,and Third Wave Technologies, Madison, Wis., are two companiesprominently involved in the development of these enzymes. ATGC'sEd Highsmith told BioWorld Today that his company is working onthe development of bacteriophage T4 resolvase and other DNAmismatch enzymes to be used in diagnostic tests for cancer andhereditary disease. n

-- Chester Bisbee Spexial To BioWorld Today

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