LONDON - Pregnant women who undergo amniocentesis to find out whether they are carrying a fetus affected by Down's syndrome or other chromosomal disorders spend up to three weeks anxiously waiting the result of the test.

Now a new test, which can provide results within a day, has been shown to be 100 percent accurate in detecting Down's syndrome. As well as speeding up the process, the new technique paves the way for tailor-made antenatal diagnosis of chromosomal disorders.

Whereas all abnormalities are reported with the current test, the latest development makes it possible for couples to specify which other chromosomal abnormalities they want examined.

Maj Hulten, professor of medical genetics in the department of biological sciences at Warwick University, told BioWorld International, “This new test is both quick and accurate. If we have the sample in the morning we can have the result by the evening. This would be a great help, because many couples find it very difficult to wait for their results. If they have been told that there is a 1 in 40 or 1 in 50 risk of something being wrong, for example, their anxiety becomes very acute when they have the amniocentesis, and a waiting time of two to three weeks for many people seems unbearable.“

Amniocentesis involves removing some of the amniotic fluid from around the fetus by inserting a needle into the uterus. This is done after about 16 weeks of pregnancy. In the U.K., about 40,000 women a year have this procedure done, because of an increased risk of having a Down's syndrome child.

For the current test, which is called karyotyping, cells shed by the fetus into the amniotic fluid are cultured and given growth factors, so that they start to divide. At a certain stage of the cell cycle, the chromosomes can be visualized. At this point, the growth of the cells is arrested, they are fixed and stained, and the chromosomes are examined through the microscope.

Although a few centers in the U.S. and U.K. are able to obtain enough cells at the correct stage of division within a week, the process usually takes an average of 15 days and sometimes as long as three weeks.

Extra Chromosome Spells Trouble

Counting the chromosomes can reveal abnormalities such as trisomy 21 (Down's syndrome, in which an extra copy of chromosome 21 is present); trisomy 18 (Edward's syndrome, in which there is an extra copy of chromosome 18); trisomy 13 (Patou's syndrome, with an extra copy of chromosome 13); and several chromosomal abnormalities involving the sex chromosomes, such as Turner's syndrome (characterized by just one copy of the X chromosome instead of XX or XY), Klinefelter's syndrome (XXY), and the XYY syndrome.

The new test for Down's syndrome, however, can be carried out on DNA extracted directly from the uncultured cells. It relies on PCR amplification of small tandem repeats (STRs) present on chromosome 21. These are repeated sequences of base pairs within the DNA, which are polymorphic: in other words, the number of repeats varies between individuals.

The technique involves adding fluorescently labeled markers for the STRs, and amplifying the STRs with PCR. This makes it possible for a machine to calculate the intensity of the fluorescence and thus, firstly, what the copy number of the STR is and, secondly, what quantity of the repeated section is present.

As Hulten explains, “Say I have 100 repeats of the STR on one of my copies of chromosome 21, and 150 repeats on the other, while my husband has 130 repeats on one of his copies of chromosome 21. If I had a child with Down's syndrome, this child may have one copy of the 100 and one of the 150 from me, as well as the third of 130 from the father.“ The new test would therefore deliver a result showing three equal peaks.

Hulten and her colleagues reported an evaluation of this method in the Lancet, July 4, 1998, in a paper titled “Rapid and simple prenatal DNA diagnosis of Down's syndrome“.

New Technique As Accurate As Standard Test

At the time the study was carried out, Hulten was clinical director of the Regional Genetics Service at Birmingham Heartlands Hospital. She and her team compared results obtained with the new technique with those obtained by traditional karyotyping. Ethical approval for the study was obtained on the grounds that neither clinicians nor patients would be informed of the results using the new technique.

Samples of amniotic fluid were used from more than 2,000 women known to be at increased risk of carrying a fetus with Down's syndrome either because of maternal age, serum testing, abnormal ultrasound scan, or they previously had a child with a chromosomal abnormality.

The researchers discovered early on it was not possible to obtain an informative result using samples of amniotic fluid which were visibly blood stained. These accounted for just 1.3 per cent of the 2,167 samples used for the study.

Of the remaining 2,139 samples, 2,083 (97.4 percent) gave an informative result when tested with two DNA markers for STRs on chromosome 21. The new test correctly identified 2,053 fetuses as normal and 30 as having Down's syndrome. These results were confirmed by traditional karyotyping.

The uninformative results occurred because both the mother and the father of these fetuses had the same number of STR copies in the markers used. This meant it was not possible to tell how many copies of chromosome 21 were present.

Hulten said, “We therefore decided to retest these samples using three markers instead of just two. Out of the 41 uninformative samples, we only had enough DNA in 33 to retest them. But when we did so, we found that, with three markers, the test would be informative for Down's syndrome in 99.6 per cent of cases. And we think that if you use four markers, it should be 100 per cent informative.“

Additional markers can be added very easily and at minimal cost, she said.

Hulten emphasized when the test is informative, it is always accurate. In addition, she said blood-stained samples still can be tested in the traditional way.

Further markers can be added to make the test diagnostic for other chromosomal abnormalities.

“My hope,“ Hulten said, “is that the nature of this new test will highlight the fact that some women and couples are not well informed when they have amniocentesis. It has become a habit with the traditional test to check for all extra chromosomes or missing chromosome segments, so that sometimes results come back which are unexpected and difficult to interpret.

“Some patients say, why did you do this test-for say Klinefelter's syndrome. They may say, 'I was supposed to be tested for Down's syndrome and now you tell me something which is very difficult to comprehend.' But the nature of the new test makes it natural to ask parents which conditions they want to be tested for.“

Another advantage is the test is easy to carry out and the results are very easy to interpret. The automated machine makes it possible to test up to 96 DNA samples at a time.

Hulten and her colleagues wrote in the Lancet: “We estimate that one scientist should be able to handle at least 5,000 samples of amniotic fluid per year, including DNA extraction, compared with the 400 cases recommended per cytogeneticist for full chromosome analysis, including cell culture.“

Although the DNA sequencing machine for carrying out PCR costs £93,000, the researchers said most regional genetics centers in the U.K. now have at least one such machine, and this may have spare capacity to run the tests for Down's syndrome.

Hulten's team is embarking on a study to compare the PCR method with fluorescence in situ hybridization (FISH), which involves targeting chromosomes 21, 18, 13, X and Y with fluorescence-labeled DNA probes that can be viewed by microscopy.

Like PCR, FISH can be carried out using uncultured cells. This two-year study, based at the University of Warwick, will compare the two techniques from several perspectives:. *

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