February 17, 2000
New Technique Greatly Increases Sensitivity of Genetic Testing
A new laboratory method improves the accuracy of current genetic
diagnostic tests for colon cancer by detecting defective genes
otherwise "masked" when one copy of the gene in question is normal. The
technique may also be used to enhance the accuracy of diagnostic tests
for a wide range of inherited diseases, including other forms of cancer
and neurological disorders.
Humans carry two copies of each gene, one of which is inherited from
the mother and one from the father. One of the weaknesses of genetic
testing is that a normal gene can mask the presence of the defective or
missing gene. In instances where masking occurs, the diagnostic tests
will either not detect the genetic defect or will prove far less
sensitive to it.
“For certain hereditary colon cancers, virtually all mutations in a gene will be detectable by this approach.”
Bert Vogelstein
"We have overcome the problem of the normal allele masking the
mutant allele by simply separating the alleles and analyzing them
independently," explained Bert
Vogelstein, a Howard Hughes Medical Institute investigator at The
Johns Hopkins University Oncology Center. In technical terms, the
technique involves conversion of the cells' paired-chromosome state of
"diploidy" to a single chromosome state of "haploidy," said
Vogelstein.
The genetic testing enhancement was reported in the February 17,
2000, Nature by researchers led by Vogelstein and Hopkins
colleagues, Hai Yan, an HHMI associate, and Kenneth Kinzler.
The conversion technique involves fusing human cells possibly
containing a defective gene with a special strain of mouse cells
developed by the researchers. The mouse cells are deficient in the
machinery that continually rejects foreign chromosomes and, thus, can
be made to harbor human genes that function normally.
Included in the assortment of fused human-mouse cells are some that
have come to possess only the single defective copy of the human gene.
The scientists can isolate these cells and use them as a basis for a
far more sensitive genetic test than using cells containing both normal
and defective gene copies.
In the Nature paper, the scientists demonstrated the
conversion technique's power by showing that they were able to verify
the presence of mutant genes in 22 patients with hereditary
non-polyposis colorectal cancer. In contrast, conventional genetic
testing detected the mutant gene in only 10 of the 22 patients.
Vogelstein and his colleagues predict that the conversion technique
will prove widely applicable to enhance tests for many other genetic
diseases.
"The sensitivity of the tests for those other diseases will depend
on the nature of the mutations," he said. "But certainly for many of
them, the sensitivity should be similarly increased substantially. And
for certain hereditary colon cancers, virtually all mutations in a gene
will be detectable by this approach.
"The technique is simple enough that it should not add much to the
costs of testing for certain diseases if it is implemented in clinical
laboratories," said Vogelstein. And in fact, in some cases conversion
will considerably lower costs by enabling more efficient new tests.
"Once you don't have the complicating diploidy and the other allele,
the signal-to-noise ratio is enhanced enormously," said Vogelstein.
"Thus, one can imagine applying a whole range of analytical techniques
now on the drawing boards that are complicated by diploidy." For
example, he said, genes from such haploid cells could be effectively
detected using "microarrays" — chips containing many thousands of
base pairs that can be tested simultaneously. Vogelstein emphasized
that the conversion technique must still be refined before it can be
introduced into routine genetic testing.
HHMI investigator Sanford Markowitz at Case Western Reserve
University, who is also an author of the Nature paper, agreed,
adding that "there is still some work to be done to make it easy to do
in clinical practice. However, the conversion strategy is a real step
forward in improving the sensitivity of these tests. The technique will
clearly increase the number of individuals for whom we will be able to
make a diagnosis." Markowitz and his colleagues are collaborating with
the Vogelstein laboratory to improve the ability to distinguish
specific cells resulting from the conversion technique.
The conversion technique will also allow basic research advances in
understanding the mechanisms of cancers and other diseases, said
Vogelstein.
"For example, there are many families with certain inherited colon
cancers in which no mutation has been found. And the question is
whether they have a mutation that conventional techniques have not
detected or whether they have a mutation in an unknown gene.
Discovering these genes would not only be important in genetic
diagnosis, but could help illuminate the mechanisms underlying the
disease," Vogelstein said.
|
