Prenatal cytogenetic diagnosis: gone FISHing, BAC soon!

Cytogenetic analysis is an important component of invasive prenatal diagnosis as chromosomal abnormalities are detected in about 1 in 200 newborns and constitute a major cause of mental retardation and congenital malformations1. Microscopic chromosome analysis of cultured cells has been regarded as the standard method for prenatal cytogenetic diagnosis since its first application to prenatal testing in 19662 and the routine use of chromosome banding analysis (karyotyping) in the early 1970s3, 4. Karyotyping has proved to be highly reliable for diagnosis of numerical chromosome abnormalities (aneuploidy) and large structural rearrangements (> 5–10 million base pairs, Mb) in fetal cells obtained invasively by either amniocentesis in the second trimester of pregnancy or chorionic villus sampling (CVS) in the first trimester since the early 1980s. The diagnostic accuracy of karyotyping cultured amniotic fluid cells has been found to be 99.4–99.8% and that of CVS 97.5–99.6%5-9. However, the main limitation of karyotyping remains the requirement for cell culture, resulting in a delay of 10–14 days for test results in many clinical genetic laboratories10. In the early 1980s, as better ultrasonographic imaging became available, less traumatic access to pure fetal blood than the fetoscopic approach was obtained from about 18 weeks' gestation by percutaneous umbilical blood sampling, also called cordocentesis11, allowing rapid karyotyping of phytohemagglutinin-stimulated lymphocytes and results within 48–72 hours. However, this procedure is generally only available at referral centers due to the need for proximity to a genetics laboratory. It is also associated with a higher risk for complications than are the other invasive tests and, hence, only performed on selected cases12. With the evolution and widespread use of screening methods and individualized risk estimates for Down syndrome based on maternal serum analytes and ultrasonographic findings13-16 and the detection of most structural abnormalities at routine fetal anomaly scan17-19 or third-trimester ultrasound20, the pattern of referral for invasive cytogenetic testing has radically changed during the last 35 years. As a result, the need for more rapid testing methods which do not require cell culture has been recognized by all parties to improve pregnancy management and alleviate parental anxiety21. Interphase fluorescence in situ hybridization (FISH), quantitative fluorescence polymerase chain reaction (QF-PCR) and several other new techniques under investigation, collectively referred to as rapid aneuploidy detection or diagnosis (RAD), have been introduced to answer specific diagnostic questions (reviewed by Shaffer and Bui22). These technologies allow rapid detection (1 or 2 days) of the most common aneuploidies: the autosomal trisomies for chromosome 13 (Patau syndrome), chromosome 18 (Edwards syndrome) and chromosome 21 (Down syndrome); aneuploidy of the sex chromosomes such as Turner syndrome (monosomy X) and Klinefelter syndrome (XXY); and triploidy, that together account for more than 80% of clinically significant chromosomal abnormalities diagnosed in the prenatal period. Mosaicism and structural rearrangements including balanced and unbalanced translocations, large deletions and duplications, and marker chromosomes account for a small part of detected cytogenetic anomalies. Although karyotyping remains the basic method, the most important technical advance in clinical cytogenetics during the last 20 years has been the development of the FISH platform. In prenatal diagnosis, different FISH applications provide increased resolution in comparison to conventional karyotyping for a comprehensive evaluation and elucidation of uncommon structural abnormalities including microdeletion syndromes, cryptic or subtle duplications and translocations, complex rearrangements and marker chromosomes22-25. The most widespread prenatal application of FISH is for rapid detection (1–2 working days) of the common aneuploidies using chromosome-specific probes applied to interphase cells from amniotic fluid and chorionic villi samples, thus obviating the delay resulting from cell culture needed for conventional cytogenetic analysis22, 24. FISH on interphase nuclei using commercially available kits has been shown in multiple studies to be a highly sensitive and specific method in the detection of aneuploidy and is a proven diagnostic application (reviewed by Shaffer and Bui22). In many experienced centers, results are now acted upon, if for example a common trisomy is found, without waiting for the examination of a complete karyotype after cell culture. Obviously, the diagnostic capability of interphase FISH is limited by the choice of probes used: uncommon structural and numerical chromosome abnormalitites other than those tested for will remain undetected. In cases of ambiguous FISH results, the possibility of karyotyping remains to resolve the diagnostic issue. An alternative approach to interphase FISH is a QF-PCR multiplex assay, which also permits the detection of major numerical chromosome disorders within 1–2 days. Highly polymorphic chromosome-specific repeat sequences (short tandem repeats (STRs) or microsatellites), which vary in length between individuals, are amplified by PCR using fluorescent primers. The products of amplification (amplicons) are visualized and quantified using an automated genetic analyzer and appropriate software. For each probe used, two peaks of fluorescence activity with a peak height or area ratio of about 1 : 1 will be produced from samples obtained from normal heterozygous fetuses (diallelic normal). Samples from trisomic fetuses will usually demonstrate either three peaks with a ratio of 1 : 1 : 1 (trisomic triallelic) or two peaks with a ratio of 2 : 1 or 1 : 2 (trisomic diallelic) for each informative probe (Figure 1). If four or more polymorphic STR markers are used for each chromosome analyzed, very few fetal samples will remain uninformative as a result of homozygosity at one locus. Electrophoretogram of QF-PCR product from an amniotic fluid sample depicting diallelic trisomy pattern with peak ratios 1 : 2 and 2 : 1 (left) and triallelic trisomy pattern with peak ratios 1 : 1 : 1 (right) consistent with trisomy 21. The Aneufast™ kit was used. AU, arbitrary unit. For more than 10 years, QF-PCR has been successfully and reliably applied to prenatal samples after both amniocentesis and CVS in many studies (reviewed by Shaffer and Bui22). QF-PCR kits are now commercially available and the accuracy of the method for the non-mosaic, common aneuploidies is similar to that of interphase FISH or karyotyping22. QF-PCR can detect mosaicism of about 20–30%26 and is superior to both traditional karyotyping and interphase FISH for the identification of maternal cell contamination27. The main advantage of QF-PCR over FISH is that it is considerably more cost-effective particularly when larger sample numbers are processed. Hence, QF-PCR is now replacing interphase FISH in an increasing number of genetics laboratories, particularly in Europe. Nevertheless, both technologies, as currently applied, have the same limitations concerning the overall detection rate of unbalanced chromosome anomalies. However, most unexpected structural cytogenetic abnormalities are relatively rare and are commonly associated with severe phenotypes that may be detected by ultrasound and thus alert the clinician and laboratory to perform additional testing beyond QF-PCR. First described in 2002, MLPA was designed to detect gene dosage abnormalities in a wide range of conditions by the relative quantification of up to 45 different DNA sequences in one reaction28, 29. The results are typically available after 2–3 days. In MLPA, it is not the nucleic acid but the probes added to the samples that are amplified and quantified29. Amplification of probes by PCR depends on the presence of small specific target sequences in the sample. Each probe produces an amplification product of unique size, which allows separation and quantification of the single fragments according to their length and fluorescence intensity by capillary electrophoresis. The number of copies of target sequence is proportional to the relative quantity of each of the PCR products. Results are given as allele copy numbers as compared to normal controls: a ratio of about 1 is obtained if both alleles are present, a ratio of about 0.5 when one allele is absent and a ratio of about 1.5 if one allele is duplicated. However, mutations or polymorphisms very close to the probe ligation site may also result in a reduced relative peak area. A MLPA kit for rapid aneuploidy detection is commercially available. Early experience of MLPA in prenatal samples is promising, but larger series need to be reported to gather more information on MLPA test performance30-32. MLPA has the same inherent limitations as those of QF-PCR in that it will not detect most structural chromosome aberrations. Moreover, maternal cell contamination and 69,XXX triploidy will not be diagnosed. However, the problem of non-informativeness of the polymorphic markers that may occur with QF-PCR is avoided altogether with MLPA. There is an ongoing debate surrounding whether RAD should be employed as an adjunct to karyotyping or whether it could be used as a stand-alone test in selected groups of women33-38. The controversy is due to the residual probability of a chromosome abnormality (both balanced and unbalanced) when RAD demonstrates a normal result. In a meta-analysis of 12 studies involving invasive tests performed for any indication, the risk of there being a chromosome aberration that was not expected to be detected by RAD methods was estimated to be 0.9%, and in 0.4% of these invasive tests the chromosome abnormality was deemed to be of clinical significance35. Similar results were found in an audit of 23 genetics laboratories in the UK34. Taken together, these series clearly show that overall in about 99% of invasive tests for chromosome analysis, women would benefit from having a RAD as it would be concordant with a karyotype analysis. However, the probability of a chromosome aberration other than the common aneuploidies is dependent on the indications for which invasive tests are being performed. Few studies have estimated the residual risk of a clinically significant chromosome aberration for different indications when RAD results are normal. In a study of 32 674 amniocenteses and CVSs performed in London, UK, mostly because of an increased risk for Down syndrome, a residual risk for a clinically significant chromosome defect of 1/1659 (0.06%) to 1/833 (0.12%) was found36. Recently, an analysis was conducted on 17 446 CVSs performed because an increased nuchal translucency was found at 11–13 weeks' gestation37. If QF-PCR analysis had been performed as a stand-alone test, 97.9% of all chromosome aberrations would have been detected. If karyotyping had been carried out only when a nuchal translucency of at least 4 mm was found and QF-PCR otherwise, only 10.1% of the cases would have needed a full karyotype whereas 99.0% of all chromosome aberrations would have been detected. This is comparable to the 97.5–99.6% accuracy of karyotyping following CVS5-9. Little is known about patients' preference. A policy has been implemented in Stockholm, Sweden since January 2005 offering the option after genetic counseling of either QF-PCR as a stand-alone analysis or a full karyotype when the indications for invasive testing are advanced maternal age (35 years or older), increased risk for a specific monogenic disorder, or parental anxiety (the expected residual risk of a clinically significant chromosome aberration is about 1 : 1000 if QF-PCR is normal based on a retrospective audit of about 14 000 invasive prenatal diagnoses, Bui et al. unpubl. observ. 2004). Experience based on over 6000 clinical samples so far shows that about 70% of Swedish women in Stockholm choose QF-PCR analysis. In array-based comparative genomic hybridization (aCGH), a comparison is made of a control genome to that of a patient's sample on an array of genomic fragments39. Currently, two main technologies are used for screening DNA copy numbers genome-wide, the BAC (Bacterial Artificial Chromosome) and the more recently developed oligonucleotide-based CGH arrays which are capable of detecting small genomic regions with amplification (often duplication) or deletion including aneuploidies39, 40. The density of the various whole-genome BAC clone sets commonly used varies from one clone per Mb to an overlapping clone set covering the entire human genome with one clone per 80 kb, while target sizes for the oligonucleotides array range from 25 to 85 bp22, 39. Thus, aCGH may address many of the limitations of both conventional karyotyping and the current RAD strategies. In contrast to the RAD methods, aCGH is a comprehensive, high-resolution, genome-wide screening strategy for obtaining DNA copy number information in a single measurement which can be rapid and less labor-intensive than karyotyping as it is readily amenable to automation. Array CGH has rapidly become an important genomic analysis tool postnatally for the identification of copy number imbalances in tumors, new microdeletion syndromes, unexplained mental retardation, and small marker chromosomes22, 39-45, and is under investigation for applications in prenatal diagnosis46-50. While currently high costs of the technology and many other issues need to be addressed before aCGH can be used routinely in prenatal diagnosis22, 51-54, it is likely to become an important tool with the potential of replacing conventional karyotyping in the future. Thus, oligonucleotides or ‘BAC to the future’55! In the longterm, non-invasive prenatal diagnosis of chromosome disorders using fetal nucleic acids circulating in maternal plasma may dramatically change the scene56, 57. For now, there are recommendations (The Genetics White Paper, UK published 2003, www.dh.gov.uk) and ethical grounds58 for rapid test results and compelling evidence22 that the vast majority of women would benefit from and prefer a rapid result after invasive cytogenetic testing, with RAD used either as an adjunct to karyotyping or as a stand-alone analysis for selected indications. It is high time to reconsider21; RAD now!