New set of probes marks chromosome tips

Rainbow of ends could facilitate automated chromosome testing

September, 1996

A transatlantic team of researchers has developed a complete set of DNA probes that can detect deletions or rearrangements at the ends of each human chromosome, where the highest concentration of genes is found. The new probes are ten times more sensitive to small defects at the chromosome ends than conventional methods, revealing abnormalities that can cause severe mental retardation or death but that are undetectable with current techniques.

The creation of the probe set was a collaborative effort between scientists at the National Institutes of Health and the Institute of Molecular Medicine at Oxford, under the leadership of David Ledbetter, PhD, now at the University of Chicago. It is reported in the September issue of Nature Genetics.

"This new set of probes will be especially valuable in genetic assessment of a child with unexplained mental retardation or for couples who have had recurrent miscarriages," said Ledbetter, professor of obstetrics and gynecology and director of the Center for Medical Genetics at the University of Chicago Medical Center. "They can uncover many of the subtle translocations or deletions that are invisible using conventional cytogenetic methods."

Conventional cytogenetic analysis relies on microscopic examination of an individual's chromosomes after they have been stained to reveal characteristic banding patterns that are unique to each chromosome. Although this method can reliably detect extra or missing chromosomes or fairly large abnormalities, it can't spot smaller "cryptic" deletions or subtle exchanges of genetic material between two chromosomes. It is also labor-intensive, expensive and slow.

Since there are fewer unique landmarks at the ends of chromosomes, conventional chromosome analysis is particularly insensitive to subtle changes in these gene-rich regions. Even at high resolution, traditional techniques can't spot deletions smaller than two-to-three-million base pairs.

Several inherited disorders, including cri-du-chat, Wolf-Hirschorn, and Miller-Dieker syndrome, have already been linked to abnormalities near the telomeres, vitally important structures at the ends of each chromosome. These disorders are difficult to detect by conventional chromosome analysis.

The new set of probes, however, all lie within 100,000 to 300,000 base pairs of the telomeres, permitting precise analysis of the integrity of each chromosome end.

In fact, while testing the probes against normal individuals, the research team serendipitously identified a family with several healthy members who carried a cryptic chromosomal translocation, a balanced exchange of genetic material between chromosomes 1 and 11.

Subsequent tests on DNA from a child from that family, who had died shortly after birth from multiple congenital anomalies, traced the infant's death to an unbalanced translocation. She had inherited from her father one normal chromosome 11 and an altered chromosome 1 - which included extra copies of genes from chromosome 11 but lacked genes normally found on chromosome 1.

The discovery allowed informative genetic counseling for this family and provides the opportunity for prenatal diagnosis in subsequent pregnancies.

Besides being more sensitive, the probe technique, known as fluorescence in-situ hybridization (FISH), is usually quicker and often less expensive than conventional chromosome studies.

FISH combines fluorescent markers with tiny bits of DNA that recognize and bind only to specific sequences of chromosomal DNA. Under fluorescent lighting, the probes emit a colored glow, usually red or green, which stands out against the light-blue-stained chromosomes.

Researchers led by Thomas Ried at the National Institutes of Health recently developed a palette of more than 20 color combinations that can be used with a series of probes to "paint" each chromosome a slightly different color.

Ledbetter and colleagues are now working to combine these multi-color techniques with their telomere probes. Currently, they administer one or two sets of probes at a time to look for abnormalities, but more extensive color coding would allow examination of more chromosomes on each pass.

"Combining multi-color discrimination with telomere probes will speed up the process and reduce the overall cost of chromosome analysis," said Ledbetter. "It would also make automated chromosome analysis a possibility, which would further cut costs."