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Prenatal diagnosis: molecular genetics and cytogenetics
Best Practice & Research Clinical Obstetrics and Gynaecology Vol. 16, No. 5, pp. 629±643, 2002
doi:10.1053/beog.2002.0327, available online at http://www.idealibrary.com on
2 Prenatal diagnosis: molecular genetics and cytogenetics The-Hung Bui
MD
Senior Consultant Clinical Geneticist and Obstetrician Gynecologist (Fetal Medicine); Director, Prenatal Diagnosis Programme and Genetic Consultation Service Department of Molecular Medicine, Clinical Genetics Unit, Karolinska Institute, Karolinska Hospital, Stockholm, Sweden Center for Fetal Medicine, Department of Obstetrics and Gynecology, Karolinska Institute, Huddinge University Hospital, Stockholm, Sweden
Elisabeth Blennow
MD, PhD
Senior Consultant Clinical Geneticist and Associate Professor
Magnus NordenskjoÈld
MD, PhD
Senior Consultant Clinical Geneticist and Professor Department of Molecular Medicine, Clinical Genetics Unit, Karolinska Institute, Karolinska Hospital, Stockholm, Sweden
The technologies developed for the Human Genome Project, the recent surge of available DNA sequences resulting from it and the increasing pace of gene discoveries and characterization have all contributed to new technical platforms that have enhanced the spectrum of disorders that can be diagnosed prenatally. The importance of determining the disease-causing mutation or the informativeness of linked genetic markers before embarking upon a DNA-based prenatal diagnosis is, however, still emphasized. Dierent ¯uorescence in situ hybridization (FISH) technologies provide increased resolution for the elucidation of structural chromosome abnormalities that cannot be resolved by more conventional cytogenetic analyses, including microdeletion syndromes, cryptic or subtle duplications and translocations, complex rearrangements involving many chromosomes, and marker chromosomes. Interphase FISH and the quantitative ¯uorescence polymerase chain reaction are ecient tools for the rapid prenatal diagnosis of selected aneuploidies, the latter being considered to be most cost-eective if analyses are performed on a large scale. There is some debate surrounding whether this approach should be employed as an adjunct to karyotyping or whether it should be used as a stand-alone test in selected groups of women. Key words: prenatal diagnosis; DNA; mutation analysis; FISH; interphase FISH; multicolour FISH; structural chromosome aberration; numerical chromosome aberration; marker chromosome; QF-PCR.
Over the past decade, new tools have been made available to clinical genetics laboratories through the technologies developed for gene-scanning, sequencing and the c 2002 Elsevier Science Ltd. All rights reserved. 1521±6934/02/$ - see front matter *
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detection of short tandem repeats (STRs) and single-nucleotide polymorphisms. The rapid pace of gene discoveries and their characterization have also been instrumental in the development of new genetic tests. Many of the major technological advances are attributable to the eorts of the international Human Genome Project, which has resulted in a high-resolution genetic map and a draft nucleotide sequence of the human genome.1,2 Thus, advances in molecular genetics and cytogenetics have provided new platforms on which to develop increased prenatal diagnostic capabilities. Pre-implantation genetic diagnosis and the particularities of mitochondrial diseases are dealt with elsewhere in this issue. This chapter focuses on current molecular and cytogenetic issues of particular relevance to obstetricians and fetal medicine specialists, and also takes a prospective view on technologies that are likely to be applied to prenatal diagnosis.
MOLECULAR GENETICS The yield of cells and DNA from a chorionic villus sample is much greater than that obtained from 20 ml amniotic ¯uid. Hence, biochemical and DNA analysis can usually be carried out directly on chorionic villi, obviating the need for and delay caused by a cell culture, as is generally required after mid-trimester amniocentesis. Many genes, for example ornithine transcarbamylase, are expressed only in specialized tissues such as the liver. Thus, biochemical diagnostic methods are limited to those diseases for which the gene products are expressed in amniocytes or chorionic villus cells, as is the case for many inherited disorders of metabolism. The ability to detect mutations at the DNA level overcomes this limitation and in principle extends diagnostic capabilities to all single-gene disorders. A very broad range of techniques is now available for the DNA-based diagnosis of hundreds of genetic disorders, a review of all these techniques and disorders being beyond the scope of this chapter. With the completion of the Human Genome Project, the number of genetic tests is expected to increase signi®cantly as a diagnostic test is one of the ®rst applications gained from knowledge about the molecular basis underlying a particular monogenic disorder.3 The human genome comprises about 28 000±35 000 genes, which code for in the region of 100 000 proteins.1,2 If only 5% of these genes have diagnostic signi®cance, approximately 1400 gene-based tests should be made available. Obviously, no laboratory can oer such a service with the current technologies as no single platform meets the breadth of these needs. In practice, whenever there are any doubts about a potentially diagnosable disorder, contact should be made with a clinical genetics unit or other specialized laboratories to enquire into the current possibility of prenatal diagnosis as the ®eld of molecular and biochemical genetics is constantly advancing. The purpose of DNA-based prenatal diagnosis is nearly always to determine whether the fetus has inherited the disease-causing mutation(s) identi®ed in one or both parents. Thus, the DNA techniques used in prenatal diagnosis are much more limited than those used when searching for unknown mutations as one is nearly always dealing either with mutation(s) previously identi®ed in an aected family member or carrier parents, or with polymorphic DNA markers known to be informative in a particular family. Ultrasound ®ndings may occasionally suggest a speci®c monogenic disorder that is amenable to molecular screening in utero, for example cystic ®brosis (echogenic fetal bowel) or thanatophoric dysplasia (very short fetal limbs).4±6
Prenatal diagnosis: molecular genetics and cytogenetics 631
Mutation spectrum and prenatal DNA analysis Several techniques are available, most of which are based upon either Southern blotting or the polymerase chain reaction (PCR). The molecular genetic techniques that are often used for prenatal diagnosis are listed in Table 1. The spectrum of mutations for commonly ordered tests varies widely, and a listing of laboratories performing speci®c tests is available from the Internet (www.geneclinics.org and www.EDDNAL.com). Most mutations are single base pair substitutions in the DNA code, leading to an altered or destroyed function of the protein encoded by a particular gene.7 Other commonly detected mutations involve short deletions or insertions of fewer than 20 nucleotides that may disrupt the reading frame of a gene (frameshift mutations) and result in the production of a nonsense protein. Less frequently, a larger disruption such as deletion or duplication of a part or all of the coding sequence of a gene occurs, as is found in about 70% of patients with Duchenne muscular dystrophy.8 Multiplex PCR, in which many parts of a gene are simultaneously ampli®ed in the same tube, is often performed for deletion screening. Triplet-repeat expansions can be detected by Southern blotting if they are very large (fragile-X syndrome or myotonic dystrophy) or by PCR (Huntington's chorea and autosomal dominant cerebellar ataxias).8 Data on reported human mutations and their associated phenotypes have been collated and are available on-line through two major databases: Online Mendelian Inheritance in Man (OMIM), maintained at the Johns Hopkins University in Baltimore, USA (www.ncbi.nlm.nih.gov/omim) and the Human Gene Mutation Database maintained at the University of Wales College of Medicine in Cardi, UK (www.hgmd.org). Most changes in the DNA do not, however, result in an abnormal protein and are known as polymorphisms. They can be used as genetic markers in the technique of linkage analysis when the mutation cannot be directly identi®ed. Many disease genes have been mapped but not yet identi®ed. In other cases, detecting the mutation can be practically impossible with the technologies used in the routine laboratory because the pattern of mutations is too variable (allele heterogeneity) or the gene too large. In these instances, linkage analysis can sometimes be used to track the mutation through the family. The importance of investigating the family by molecular genetic testing before embarking upon a DNA-based prenatal diagnosis cannot be overemphasized. Patients considering prenatal diagnosis should be advised to seek genetic counselling and testing before becoming pregnant as only then can they start a pregnancy in the full knowledge of all the options available to them. Table 1. Molecular genetic techniques commonly used for prenatal diagnosis. Detection of known point mutations, splicing mutations, small insertions or deletions . PCR ampli®cation and restriction enzyme analysis to detect mutations that alter restriction sites . PCR ampli®cation and allele-speci®c oligonucleotide hybridization . PCR ampli®cation and direct automated sequencing Detection of large insertions, triplet-repeat expansions, deletions and other large structural rearrangements . Southern blot analysis . PCR ampli®cation, multiplex PCR Linkage-based analysis using intragenic DNA polymorphisms PCR, polymerase chain reaction.
632 T.-H. Bui, E. Blennow and M. NordenskjoÈld
The causative mutation must be identi®ed in the index case or, whenever possible, in the parents. The family should be investigated as early as possible to save time and to con®rm diagnosis. As the work-up of a family can take several weeks, it is often too late to begin such work-up when a woman is already advanced in her pregnancy. Much DNA testing is sequential; ®rst, the most common mutations will be looked for, screening for rarer mutations following if the ®rst test is negative. Many mutations in dierent genes produce the same phenotype (locus heterogeneity). Prenatal diagnosis is not possible in such cases unless the test to be applied has been shown to be applicable to a particular family. This will usually involve demonstrating that a mutation is present in an aected family member. Similarly, it is essential to determine which DNA markers are informative before a linkage-based prenatal diagnosis is oered. Preparation for linkage analysis can involve the investigation of many family members with dierent polymorphic markers before informative ¯anking or intragenic markers are found. CYTOGENETICS Although karyotyping remains the gold standard of chromosome analysis and still is the most frequently used genetic method in prenatal diagnosis, the most important advance in cytogenetics during the past 20 years has been the development of ¯uorescence in situ hybridization (FISH) technologies. Thorough accounts of molecular cytogenetic methods have recently been published in this series and elsewhere, and will therefore only be brie¯y reviewed here.9±11 Fluorescence in situ hybridization FISH makes use of speci®c DNA probes, labelled by incorporating chemically modi®ed nucleotides that ¯uoresce directly or can be detected by binding a ¯uorescently tagged reporter molecule.9 These single-stranded probes may be hybridized not only to metaphase chromosome spreads, but also directly to the ®xed interphase nuclei of non-dividing cells. FISH probes can detect regions as small as 0.5 kb on metaphase chromosomes.12 As a comparison, the smallest chromosome abnormality detectable is about 2000±3000 kb when banding techniques are used on highly extended chromosomes in pro-metaphase. Major technical developments have included the ability to generate a wide variety of probes as well as improvements in digital imaging microscopy and software.9±12 Four main classes of FISH probe are available: locus-speci®c probes, repetitive-sequence probes (alphoid or centromeric probes, telomeric probes), whole-genomic DNA probes and chromosome-speci®c painting probes.12 A complete and optimized set of probes for the simultaneous FISH analysis of the subtelomeric regions of all human chromosomes is now available.13 This technology allows the detection of subtle deletions, duplications and translocations involving telomeres and subtelomeric regions, except for the short arms of the acrocentric chromosomes, which contain shared repetitive sequences and satellite DNA. Deletions or duplications of these short arms have no phenotypic consequences. Furthermore, several systems for multicolour FISH have, over the past few years, been developed and applied to karyotyping and the detection of chromosomal abnormalities. Spectral karyotyping (SKY), which uses Fourier spectroscopy and charged couple device imaging to measure emission spectra (Figure 1)12±16, and multiplex FISH (M-FISH)17,18
Prenatal diagnosis: molecular genetics and cytogenetics 633
Figure 1. G-banded karyotype with a suspicious subtle balanced reciprocal translocation between segments on the distal long arm of chromosome 7 and 10 (left). Spectral karyotyping (SKY) of the same case con®rming without any doubt the diagnosis of t(7;10)(q36;q26).
are based on the simultaneous hybridization of 24 chromosome-speci®c painting probes labelled with dierent combinations of ®ve ¯uorochromes. In combined binary ratio labelling FISH (COBRA FISH),19 the discrimination of chromosomes is based on the ratio of the hybridization signal intensities of three or four ¯uorochromes, whereas in colour-changing karyotyping, it is based on the combinatorial labelling of chromosomes.20 All these multicolour systems can detect numerical and interchromosomal aberrations in a single hybridization, but intrachromosomal aberrations, such as paracentric or pericentric inversions, small deletions and duplications, remain impossible to identify. This is resolved by the most recent re®nements of FISH, which include multicolour chromosome banding, based on the use of dierently labelled, overlapping microdissection libraries,21,22 and the simultaneous dierential painting of each chromosome arm apart from the short arms of the acrocentric chromosomes.23 Another notable enhancement is centromere-speci®c multicolour FISH (cenM-FISH), which distinguishes all the centromeric regions except those of the evolutionary highly conserved chromosomes 13 and 21.24 The increased resolution provided by dierent FISH technologies, alone or combined, has been shown in numerous studies of structural chromosome abnormalities. These include microdeletion syndromes, cryptic duplications or translocations, complex rearrangements and marker chromosomes.9±13,15±16,18±19,22,24±25 In prenatal diagnosis, dierent FISH technologies have been applied to identify and characterize structural chromosome abnormalities that cannot be resolved or diagnosed by conventional chromosome banding techniques.
Presence of a marker chromosome The identi®cation of marker chromosomes during prenatal chromosome analysis raises serious concerns regarding the phenotypic consequences to the fetus and can create dicult counselling problems.26±28 These small chromosome markers are structurally abnormal and cannot be identi®ed with conventional chromosome banding techniques. In order to avoid ambiguities, the terms `structurally abnormal chromosome' and `extra structurally abnormal chromosome' (ESAC) have been coined. The terms ESAC and `supernumerary marker chromosome' are synonymous.
634 T.-H. Bui, E. Blennow and M. NordenskjoÈld
The incidence of ESAC has been estimated to be 0.2±0.72 per 1000 newborn infants. The prevalence of marker chromosomes found in larger prenatal series also varies considerably, with a reported range of 0.6±1.5 per 1000.26 The chromosomal origin and composition of ESACs are directly associated to their phenotypic presentation. ESACs that are well-established chromosomal syndromes include isochromosome 9p, isochromosome 12p mosaicism (Pallister±Killian syndrome), isochromosome 18p and the inverted duplications inv(15) and dup(22) (cat eye syndrome). These ESACs can generally be diagnosed or suggested by conventional chromosome banding techniques. FISH is only used to con®rm the diagnosis when some doubt remains. The introduction of FISH methods in the early 1990s using chromosome-, locus- or centromere-speci®c probes has provided a powerful approach to the identi®cation of ESACs (reviewed in reference 29). Methods for de®ning ESACs are now fairly well established.27,29 Previously, each probe (or a combination of a few probes) was tried in turn to de®ne the ESAC, making the whole process highly inecient and time-consuming.30 Reverse painting using degenerate oligonucleotide primed-PCR libraries is the most informative way to outline an ESAC.31 This can be achieved by isolating the ESAC by ¯ow sorting or microdissection and using it as a template to generate an ESAC-speci®c library by PCR.31±33 Not only does this reveal the origin of the chromosome(s) involved, but it also pinpoints the precise chromosomal segments that are contained within the marker (Figure 2). When reverse painting is not available, M-FISH or SKY will, in most cases, elucidate the origin of the ESAC in a single hybridization.12,18 The cenM-FISH technique is particularly helpful for the characterization of small ESACs with no (or nearly no) euchromatin when, in addition, the amount of available sample material is restricted, as in prenatal cases.24 The most common group of ESACs involves only the paracentromeric region of a chromosome, often with satellites at one or both ends. Using FISH, about 80% of ESACs have been shown to be derived from the acrocentric chromosomes. Many of these are either pseudo idic(15), also called inverted duplication, inv dup(15), or ESACs derived from chromosome 22.26,34 When the inv dup(15) includes the Prader±Willi/ Angelman critical region, it is associated with mild-to-severe mental retardation and other pathological features.35,36 Chromosome 22 ESACs without euchromatin are not associated with an adverse phenotype. Rare analphoid marker chromosomes with a neocentromere have been reported (reviewed in reference 37). About 40% of ESACs found on prenatal analysis are inherited, and the risk of an adverse phenotype in the fetus is usually low if the carrier parent has a normal clinical phenotype. The residual risk results from the possibility of uniparental disomy38, i.e. the inheritance of both homologous chromosomes from only one parent, or the fact that the fetus has inherited an ESAC in a non-mosaic form from a mosaic parent. A review of de novo cases of non-acrocentric, autosomal ESACs characterized by FISH gave a risk estimate of an abnormal phenotype of about 28%.27,28 ESACs derived from acrocentric chromosome may result in a risk comparable to that of the other autosomes if de novo supernumerary marker chromosomes derived from chromosomes 15 and 22 ± of which the majority are clinically normal ± are excluded. The unequivocal identi®cation of the origin and composition of ESACs is clearly crucial for karyotype±phenotype correlation and a more accurate assessment of risk in individual cases. Ultrasound examination for fetal anomalies may also assist in this individual assessment.27
Prenatal diagnosis: molecular genetics and cytogenetics 635
Figure 2. (A) Fluorescence in situ hybridization (FISH) on a metaphase chromosome spread with a chromosome 8-speci®c painting probe, showing the two normal chromosomes 8 (red colour) and the origin of a small ring marker. (B) Microdissection of the supernumerary ring marker chromosome that will be used as a template to generate a marker-speci®c library by a polymerase chain reaction. (C) FISH using the marker-speci®c probe reveals not only the origin of the ring marker chromosome, but also the chromosomal segments involved and the ®nal karyotype: 47,XX,r(8)(q10q21.1)de novo.
RAPID PRENATAL DIAGNOSIS OF THE MOST COMMON NUMERICAL CHROMOSOME ABNORMALITIES Two methods have recently been introduced for the rapid prenatal diagnosis of numerical chromosome abnormalities. These are interphase FISH and quantitative ¯uorescent polymerase chain reaction (QF-PCR) assay. Interphase FISH The most widespread application of FISH in prenatal diagnosis is for the rapid detection (1±2 working days) of the common numerical chromosome abnormalities using chromosome-speci®c probes applied to interphase cells from amniocenteses and chorionic villi samples (Figure 3), thus obviating the 7±14 days' delay resulting from cell culture associated with conventional chromosome analysis.39±42 Most commonly,
Figure 3. Interphase ¯uorescence in situ hybridization on uncultured amniocytes. (A) Three green signals for a chromosome 13-speci®c probe (there being in one nucleus a partial masking of one red signal by one green signal). Thus, the fetus carries a trisomy 13. (B) Three signals for each of the chromosome-speci®c probes used in three nuclei from the same amniotic ¯uid sample. Left, three green signals for a chromosome 13 probe; middle, three red signals each for chromosome 13 probe and three signals for the chromosome 21 probe; right, three light blue signals for the chromosome 18 probe. The fetus therefore has a triploidy.
636 T.-H. Bui, E. Blennow and M. NordenskjoÈld
Prenatal diagnosis: molecular genetics and cytogenetics 637
probes speci®c for chromosomes 13, 18, 21, X and Y are used as, depending on the indications for invasive tests, numerical abnormalities involving these chromosomes account for 70±95% of the chromosome aberrations identi®ed prenatally.9±11,39±43 The usefulness of interphase FISH in prenatal diagnosis and the test performance characteristics have been recently reviewed in 29 039 informative testing events using one commercially available assay (AneuVision, Vysis Inc, Downers Grove, IL, USA) and 18 275 other specimens tested with a diversity of other probes.39 With the AneuVision assay, the detection rate was nearly 100%, with a false-positive rate of only 0.003% and a false-negative rate of 0.024%. In many centres with a wide experience of interphase FISH, results are now acted upon if, for example, a common trisomy is found, without waiting for the examination of a complete karyotype after cytoculture. The diagnostic capability of interphase FISH is obviously limited by the choice of probe used: structural chromosome aberrations and numerical chromosome abnormalities other than those tested for will be missed. In addition, the incomplete hybridization of FISH probes or a reduction in signal size due to heteromorphisms39,44±47 may lead to falsenegative prenatal FISH results. More recent FISH probe sets use locus-speci®c probes that avoid the problems of centromeric heteromorphisms. In cases of ambiguous FISH results, however, full karyotyping should remain the gold standard. Recently, chromosome arm-speci®c subtelomere probes have been used successfully to rapidly determine the segregation of a parental balanced reciprocal translocation in uncultured amniocytes,48,49 an approach similar to that used in pre-implantation genetic diagnosis on single cells (see Chapter 4 in this issue). Chromosome arm-speci®c pro-telomere probes are available for each chromosome,13 and it would be possible to oer a rapid diagnosis for similar indications in selected couples.
Quantitative ¯uorescent polymerase chain reaction An alternative approach to interphase FISH is to employ a QF-PCR assay, which also allows the detection of major numerical chromosome disorders within a few hours of sample collection. Highly polymorphic chromosome-speci®c STRs are ampli®ed by a PCR. A ¯uorochrome incorporated into the products of ampli®cation permits visualization and quanti®cation using an automated DNA scanner. Two peaks of ¯uorescence activity with a ratio of about 1:1 will be produced for each chromosomespeci®c STR from samples obtained from normal heterozygous fetuses. 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 (trisomic diallelic). The principle of the QF-PCR test using STRs is illustrated in Figure 4.50 If three or more polymorphic STR markers are used for each chromosome analysed, very few fetal samples will remain uninformative as a result of homozygosity at one locus. In recent years, QF-PCR has been successfully applied to determine the copy number of ®rst chromosomes 13, 18, 21 (Figure 5) and later chromosomes X and Y in uncultured amniocytes or chorionic villi for prenatal diagnosis (reviewed in reference 50).51±57 The advantage of QF-PCR over FISH is that it is considered substantially more cost-eective, especially when larger sample numbers are processed: less material is required, bloody specimens may be successfully processed, and QF-PCR is less time-consuming and labour intensive. However, the same limitations concerning the overall detection rate of unbalanced chromosome anomalies apply for both techniques as currently applied.
638 T.-H. Bui, E. Blennow and M. NordenskjoÈld
Figure 4. Principle underlying the quantitative ¯uorescence polymerase chain reaction for the rapid prenatal diagnosis of common aneuploidies. Reproduced with permission from Adinol® et al (2000).50
FUTURE PROSPECTS AND CONCLUSIONS Over the past decade, an impressive array of technologies has emerged to analyse variant DNA and structure in the human genome. Many of these methods have now been incorporated into the arsenal available to routine molecular genetics laboratories. DNAbased prenatal diagnosis is currently targeted towards families at risk of an inherited condition and is not used as a screening test because of cost, technical complexity and limited resources. In the future, an increasing number of single-gene disorders will be amenable to testing from chorion villus cells or amniocytes and also potentially from fetal cells isolated from the maternal circulation or fetal DNA in maternal plasma.58±59 New analytical devices including comparative genome hybridization11 and microchips60
Prenatal diagnosis: molecular genetics and cytogenetics 639
Figure 5. Partial electrophoretograms of the quantitative ¯uorescent polymerase chain reaction products of dierent chromosome 21-speci®c short tandem repeat markers. (A) Normal disomic heterozygote (ratio 1:1). (B) Trisomic triallelic (ratio 1:1:1) for marker D21S226 and trisomic diallelic (ratio 2:1) for marker IFNAR. The fetus thus has trisomy 21. (C) Trisomic triallelic (ratio 1:1:1), indicating a fetus with trisomy 21. Reproduced with permission from Dr HeleÂne Olsson, CyberGene AB, Huddinge, Sweden.
640 T.-H. Bui, E. Blennow and M. NordenskjoÈld
are being applied to molecular genetics and are likely to ®nd applications in clinical medicine. Eorts to develop better methods for target probe and signal ampli®cation will result in better DNA detection tools.61 The introduction of multicolour and other FISH technologies has contributed signi®cantly to the detailed characterization of cryptic or subtle structural chromosome abnormalities, complex rearrangements and ESACs in prenatal diagnosis, as well as providing a better understanding of their clinical signi®cance. Both interphase FISH and QF-PCR are ecient tools for the rapid prenatal diagnosis of common aneuploidies, although current experience suggests that QF-PCR is more cost-eective in large-scale application. Whether QF-PCR should be performed as an adjunct to full karyotyping or be used as a stand-alone test, at least for some select indications, is currently under debate. Acknowledgement Supported in part by the European Commission (ENFET concerted action).
Practice points . an extensive genetic work-up and counselling are needed before DNA-based prenatal diagnosis can be oered for selected single-gene disorders . karyotyping remains the `gold standard' in prenatal diagnosis . dierent FISH technologies provide increased resolution and allow the de®nition of structural chromosome abnormalities that cannot be resolved by conventional chromosome analysis . both interphase FISH and QF-PCR allow the rapid (1±2 day) prenatal diagnosis of common aneuploidies . QF-PCR appears to be more cost-eective for large-scale use
Research agenda increased pace in the discovery and characterization of disease-causing genes further development in technologies for mutation analysis and FISH further studies on fetal DNA and cells in the maternal circulation further large-scale clinical studies of QF-PCR for the rapid prenatal diagnosis of selected numerical abnormalities . better methods for the ampli®cation of target, probes and signals for DNA detection tools . . . .
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American Journal of Medical Genetics 1993; 53: 433±442. *31. Anderlid BM, Sahlen S, Schoumans J et al. Detailed characterization of 12 supernumerary ring chromosomes using micro-FISH and search for uniparental disomy. American Journal of Medical Genetics 2001; 99: 223±233.
642 T.-H. Bui, E. Blennow and M. NordenskjoÈld 32. Blennow E, Telenius H, Larsson C et al. Complete characterization of a large marker chromosome by reverse and forward chromosome painting. Human Genetics 1992; 90: 371±374. 33. Xu J, Fong CT, Cedrone E et al. Prenatal identi®cation of de novo marker chromosomes using microFISH approach. Clinical Genetics 1998; 53: 490±496. 34. Blennow E, Brodum-Nielsen K, Telenius H et al. Fifty probands with extra structurally abnormal chromosomes characterized by ¯uorescence in situ hybridization. American Journal of Medical Genetics 1995; 55: 85±94. 35. Mignon C, Malzac P, Moncla A et al. Clinical heterogeneity in 16 patients with inv dup 15 chromosomes: cytogenetic and molecular studies, search for an imprinting eect. European Journal of Human Genetics 1996; 4: 88±100. 36. Cotter PD, Ledesma CT, Dietz LG et al. Prenatal diagnosis of supernumerary marker 15 chromosomes and exclusion of uniparental disomy for chromosome 15. Prenatal Diagnosis 1999; 19: 721±726. 37. Reddy KS, Sulcova V, Schwartz S et al. Mosaic tetrasomy 8q: inverted duplication of 8q23.3qter in an analphoid marker. American Journal of Medical Genetics 2000; 92: 69±76. 38. Kotzot D. Complex and segmental uniparental disomy (UPD): review and lessons from rare chromosomal complements. Journal of Medical Genetics 2001; 38: 497±507. *39. Tepperberg J, Pettenati MJ, Rao PN et al. Prenatal diagnosis using interphase ¯uorescence in situ hybridization (FISH): 2-year multi-center retrospective study and review of the literature. Prenatal Diagnosis 2001; 21: 293±301. 40. Sawa R, Hayashi Z, Tanaka T et al. Rapid detection of chromosome aneuploidies by prenatal interphase FISH (¯uorescence in situ hybridization) and its clinical utility in Japan. Journal of Obstetrical and Gynaecological Research 2001; 27: 41±47. 41. Cheong Leung W, Chitayat D, Seaward G et al. Role of amniotic ¯uid interphase ¯uorescence in situ hybridization (FISH) analysis in patient management. Prenatal Diagnosis 2001; 21: 327±332. 42. Witters I, Devriendt K, Legius E et al. Rapid prenatal diagnosis of trisomy 21 in 5049 consecutive uncultured amniotic ¯uid samples by ¯uorescence in situ hybridisation (FISH). Prenatal Diagnosis 2002; 22: 29±33. 43. Evans MI, Henry GP, Miller WA et al. International collaborative assessment of 146,000 prenatal karyotypes: expected limitations if only chromosome-speci®c probes and ¯uorescent in situ hybridization are used. Human Reproduction 1999; 14: 1213±1216. 44. Thilaganathan B, Sairam S, Ballard T et al. Eectiveness of prenatal chromosomal analysis using multicolor ¯uorescent in situ hybridization. British Journal of Obstetrics and Gynaecology 2000; 107: 262±266. 45. Verma RS, Batish SD, Gogineni SK et al. Centromeric alphoid DNA heteromorphisms of chromosome 21 revealed by FISH-technique. Clinical Genetics 1997; 51: 91±93. 46. Verma RS, Ishwar L, Gogineni SK et al. Pericentromeric heteromorphism of human chromosome 18 as revealed by FISH-technique. Annales de GeÂneÂtique 1998; 41: 154±156. 47. Weremowicz S, Sandstrom DJ, Morton CC et al. Fluorescence in situ hybridization (FISH) for rapid detection of aneuploidy: experience with 911 prenatal cases. Prenatal Diagnosis 2001; 21: 262±269. 48. Cotter PD & Musci TJ. Interphase FISH with chromosome-speci®c protelomere probes for rapid prenatal diagnosis in a reciprocal translocation carrier. Prenatal Diagnosis 2001; 21: 171±175. 49. Pettenati MJ, Von Kap-Herr C, Jackle B et al. Rapid interphase analysis for prenatal diagnosis of translocation carriers using subtelomeric probes. Prenatal Diagnosis 2002; 22: 193±197. *50. Adinol® M, Sherlock J, Cirigliano V et al. Prenatal screening for aneuploidies by quantitative ¯uorescent polymerase chain reaction. Community Genetics 2000; 3: 50±60. 51. Bili C, Divane A, Apessos A et al. Prenatal diagnosis of common aneuploidies using quantitative ¯uorescent PCR. Prenatal Diagnosis 2002; 22: 360±365. 52. Cioni R, Bussani C, Scarselli B et al. Detection of fetal cells in intrauterine lavage samples collected in the ®rst trimester of pregnancy. Prenatal Diagnosis 2000; 22: 52±55. 53. Mann K, Fox SP, Abbs SJ et al. Development and implementation of a new rapid aneuploidy diagnostic service within the UK National Health Service and implications for the future of prenatal diagnosis. Lancet 2001; 358: 1057±1061. 54. Adinol® M & Sherlock J. Prenatal detection of chromosome disorders by QF-PCR. Lancet 2001; 358: 1030±1031. 55. Cirigliano V, Ejarque M, Canadas MP et al. Clinical application of multiplex quantitative ¯uorescent polymerase chain reaction (QF-PCR) for the rapid prenatal detection of common chromosome aneuploidies. Molecular Human Reproduction 2001; 7: 1001±1006. 56. Jenderny J, Schmidt W, Hecher K et al. Increased nuchal translucency, hydrops fetalis or hygroma colli. A new test strategy for early fetal aneuploidy detection. Fetal Diagnosis and Therapy 2001; 16: 211±214. 57. Schmidt W, Jenderny J, Hecher K et al. Detection of aneuploidy in chromosomes X, Y, 13, 18 and 21 by QF-PCR in 662 selected pregnancies at risk. Molecular Human Reproduction 2000; 6: 855±860.
Prenatal diagnosis: molecular genetics and cytogenetics 643 58. Holzgreve W. Fetal cells in cervical mucus and maternal blood. BaillieÁre's Clinical Obstetrics and Gynaecology 2000; 14: 709±722. *59. Lo YM. Circulating nucleic acids in plasma and serum: an overview. Annals of the New York Academy of Sciences 2001; 945: 1±7. 60. Cheng J, Fortina P, Surrey S et al. Microchip-based devices for molecular diagnosis of genetic diseases. Molecular Diagnosis 1996; 1: 183±200. 61. Andras SC, Power JB, Cocking EC & Davey MR. Strategies for signal ampli®cation in nucleic acid detection. Molecular Biotechnology 2001; 19: 29±44.
doi:10.1053/beog.2002.0327, available online at http://www.idealibrary.com on
2 Prenatal diagnosis: molecular genetics and cytogenetics The-Hung Bui
MD
Senior Consultant Clinical Geneticist and Obstetrician Gynecologist (Fetal Medicine); Director, Prenatal Diagnosis Programme and Genetic Consultation Service Department of Molecular Medicine, Clinical Genetics Unit, Karolinska Institute, Karolinska Hospital, Stockholm, Sweden Center for Fetal Medicine, Department of Obstetrics and Gynecology, Karolinska Institute, Huddinge University Hospital, Stockholm, Sweden
Elisabeth Blennow
MD, PhD
Senior Consultant Clinical Geneticist and Associate Professor
Magnus NordenskjoÈld
MD, PhD
Senior Consultant Clinical Geneticist and Professor Department of Molecular Medicine, Clinical Genetics Unit, Karolinska Institute, Karolinska Hospital, Stockholm, Sweden
The technologies developed for the Human Genome Project, the recent surge of available DNA sequences resulting from it and the increasing pace of gene discoveries and characterization have all contributed to new technical platforms that have enhanced the spectrum of disorders that can be diagnosed prenatally. The importance of determining the disease-causing mutation or the informativeness of linked genetic markers before embarking upon a DNA-based prenatal diagnosis is, however, still emphasized. Dierent ¯uorescence in situ hybridization (FISH) technologies provide increased resolution for the elucidation of structural chromosome abnormalities that cannot be resolved by more conventional cytogenetic analyses, including microdeletion syndromes, cryptic or subtle duplications and translocations, complex rearrangements involving many chromosomes, and marker chromosomes. Interphase FISH and the quantitative ¯uorescence polymerase chain reaction are ecient tools for the rapid prenatal diagnosis of selected aneuploidies, the latter being considered to be most cost-eective if analyses are performed on a large scale. There is some debate surrounding whether this approach should be employed as an adjunct to karyotyping or whether it should be used as a stand-alone test in selected groups of women. Key words: prenatal diagnosis; DNA; mutation analysis; FISH; interphase FISH; multicolour FISH; structural chromosome aberration; numerical chromosome aberration; marker chromosome; QF-PCR.
Over the past decade, new tools have been made available to clinical genetics laboratories through the technologies developed for gene-scanning, sequencing and the c 2002 Elsevier Science Ltd. All rights reserved. 1521±6934/02/$ - see front matter *
630 T.-H. Bui, E. Blennow and M. NordenskjoÈld
detection of short tandem repeats (STRs) and single-nucleotide polymorphisms. The rapid pace of gene discoveries and their characterization have also been instrumental in the development of new genetic tests. Many of the major technological advances are attributable to the eorts of the international Human Genome Project, which has resulted in a high-resolution genetic map and a draft nucleotide sequence of the human genome.1,2 Thus, advances in molecular genetics and cytogenetics have provided new platforms on which to develop increased prenatal diagnostic capabilities. Pre-implantation genetic diagnosis and the particularities of mitochondrial diseases are dealt with elsewhere in this issue. This chapter focuses on current molecular and cytogenetic issues of particular relevance to obstetricians and fetal medicine specialists, and also takes a prospective view on technologies that are likely to be applied to prenatal diagnosis.
MOLECULAR GENETICS The yield of cells and DNA from a chorionic villus sample is much greater than that obtained from 20 ml amniotic ¯uid. Hence, biochemical and DNA analysis can usually be carried out directly on chorionic villi, obviating the need for and delay caused by a cell culture, as is generally required after mid-trimester amniocentesis. Many genes, for example ornithine transcarbamylase, are expressed only in specialized tissues such as the liver. Thus, biochemical diagnostic methods are limited to those diseases for which the gene products are expressed in amniocytes or chorionic villus cells, as is the case for many inherited disorders of metabolism. The ability to detect mutations at the DNA level overcomes this limitation and in principle extends diagnostic capabilities to all single-gene disorders. A very broad range of techniques is now available for the DNA-based diagnosis of hundreds of genetic disorders, a review of all these techniques and disorders being beyond the scope of this chapter. With the completion of the Human Genome Project, the number of genetic tests is expected to increase signi®cantly as a diagnostic test is one of the ®rst applications gained from knowledge about the molecular basis underlying a particular monogenic disorder.3 The human genome comprises about 28 000±35 000 genes, which code for in the region of 100 000 proteins.1,2 If only 5% of these genes have diagnostic signi®cance, approximately 1400 gene-based tests should be made available. Obviously, no laboratory can oer such a service with the current technologies as no single platform meets the breadth of these needs. In practice, whenever there are any doubts about a potentially diagnosable disorder, contact should be made with a clinical genetics unit or other specialized laboratories to enquire into the current possibility of prenatal diagnosis as the ®eld of molecular and biochemical genetics is constantly advancing. The purpose of DNA-based prenatal diagnosis is nearly always to determine whether the fetus has inherited the disease-causing mutation(s) identi®ed in one or both parents. Thus, the DNA techniques used in prenatal diagnosis are much more limited than those used when searching for unknown mutations as one is nearly always dealing either with mutation(s) previously identi®ed in an aected family member or carrier parents, or with polymorphic DNA markers known to be informative in a particular family. Ultrasound ®ndings may occasionally suggest a speci®c monogenic disorder that is amenable to molecular screening in utero, for example cystic ®brosis (echogenic fetal bowel) or thanatophoric dysplasia (very short fetal limbs).4±6
Prenatal diagnosis: molecular genetics and cytogenetics 631
Mutation spectrum and prenatal DNA analysis Several techniques are available, most of which are based upon either Southern blotting or the polymerase chain reaction (PCR). The molecular genetic techniques that are often used for prenatal diagnosis are listed in Table 1. The spectrum of mutations for commonly ordered tests varies widely, and a listing of laboratories performing speci®c tests is available from the Internet (www.geneclinics.org and www.EDDNAL.com). Most mutations are single base pair substitutions in the DNA code, leading to an altered or destroyed function of the protein encoded by a particular gene.7 Other commonly detected mutations involve short deletions or insertions of fewer than 20 nucleotides that may disrupt the reading frame of a gene (frameshift mutations) and result in the production of a nonsense protein. Less frequently, a larger disruption such as deletion or duplication of a part or all of the coding sequence of a gene occurs, as is found in about 70% of patients with Duchenne muscular dystrophy.8 Multiplex PCR, in which many parts of a gene are simultaneously ampli®ed in the same tube, is often performed for deletion screening. Triplet-repeat expansions can be detected by Southern blotting if they are very large (fragile-X syndrome or myotonic dystrophy) or by PCR (Huntington's chorea and autosomal dominant cerebellar ataxias).8 Data on reported human mutations and their associated phenotypes have been collated and are available on-line through two major databases: Online Mendelian Inheritance in Man (OMIM), maintained at the Johns Hopkins University in Baltimore, USA (www.ncbi.nlm.nih.gov/omim) and the Human Gene Mutation Database maintained at the University of Wales College of Medicine in Cardi, UK (www.hgmd.org). Most changes in the DNA do not, however, result in an abnormal protein and are known as polymorphisms. They can be used as genetic markers in the technique of linkage analysis when the mutation cannot be directly identi®ed. Many disease genes have been mapped but not yet identi®ed. In other cases, detecting the mutation can be practically impossible with the technologies used in the routine laboratory because the pattern of mutations is too variable (allele heterogeneity) or the gene too large. In these instances, linkage analysis can sometimes be used to track the mutation through the family. The importance of investigating the family by molecular genetic testing before embarking upon a DNA-based prenatal diagnosis cannot be overemphasized. Patients considering prenatal diagnosis should be advised to seek genetic counselling and testing before becoming pregnant as only then can they start a pregnancy in the full knowledge of all the options available to them. Table 1. Molecular genetic techniques commonly used for prenatal diagnosis. Detection of known point mutations, splicing mutations, small insertions or deletions . PCR ampli®cation and restriction enzyme analysis to detect mutations that alter restriction sites . PCR ampli®cation and allele-speci®c oligonucleotide hybridization . PCR ampli®cation and direct automated sequencing Detection of large insertions, triplet-repeat expansions, deletions and other large structural rearrangements . Southern blot analysis . PCR ampli®cation, multiplex PCR Linkage-based analysis using intragenic DNA polymorphisms PCR, polymerase chain reaction.
632 T.-H. Bui, E. Blennow and M. NordenskjoÈld
The causative mutation must be identi®ed in the index case or, whenever possible, in the parents. The family should be investigated as early as possible to save time and to con®rm diagnosis. As the work-up of a family can take several weeks, it is often too late to begin such work-up when a woman is already advanced in her pregnancy. Much DNA testing is sequential; ®rst, the most common mutations will be looked for, screening for rarer mutations following if the ®rst test is negative. Many mutations in dierent genes produce the same phenotype (locus heterogeneity). Prenatal diagnosis is not possible in such cases unless the test to be applied has been shown to be applicable to a particular family. This will usually involve demonstrating that a mutation is present in an aected family member. Similarly, it is essential to determine which DNA markers are informative before a linkage-based prenatal diagnosis is oered. Preparation for linkage analysis can involve the investigation of many family members with dierent polymorphic markers before informative ¯anking or intragenic markers are found. CYTOGENETICS Although karyotyping remains the gold standard of chromosome analysis and still is the most frequently used genetic method in prenatal diagnosis, the most important advance in cytogenetics during the past 20 years has been the development of ¯uorescence in situ hybridization (FISH) technologies. Thorough accounts of molecular cytogenetic methods have recently been published in this series and elsewhere, and will therefore only be brie¯y reviewed here.9±11 Fluorescence in situ hybridization FISH makes use of speci®c DNA probes, labelled by incorporating chemically modi®ed nucleotides that ¯uoresce directly or can be detected by binding a ¯uorescently tagged reporter molecule.9 These single-stranded probes may be hybridized not only to metaphase chromosome spreads, but also directly to the ®xed interphase nuclei of non-dividing cells. FISH probes can detect regions as small as 0.5 kb on metaphase chromosomes.12 As a comparison, the smallest chromosome abnormality detectable is about 2000±3000 kb when banding techniques are used on highly extended chromosomes in pro-metaphase. Major technical developments have included the ability to generate a wide variety of probes as well as improvements in digital imaging microscopy and software.9±12 Four main classes of FISH probe are available: locus-speci®c probes, repetitive-sequence probes (alphoid or centromeric probes, telomeric probes), whole-genomic DNA probes and chromosome-speci®c painting probes.12 A complete and optimized set of probes for the simultaneous FISH analysis of the subtelomeric regions of all human chromosomes is now available.13 This technology allows the detection of subtle deletions, duplications and translocations involving telomeres and subtelomeric regions, except for the short arms of the acrocentric chromosomes, which contain shared repetitive sequences and satellite DNA. Deletions or duplications of these short arms have no phenotypic consequences. Furthermore, several systems for multicolour FISH have, over the past few years, been developed and applied to karyotyping and the detection of chromosomal abnormalities. Spectral karyotyping (SKY), which uses Fourier spectroscopy and charged couple device imaging to measure emission spectra (Figure 1)12±16, and multiplex FISH (M-FISH)17,18
Prenatal diagnosis: molecular genetics and cytogenetics 633
Figure 1. G-banded karyotype with a suspicious subtle balanced reciprocal translocation between segments on the distal long arm of chromosome 7 and 10 (left). Spectral karyotyping (SKY) of the same case con®rming without any doubt the diagnosis of t(7;10)(q36;q26).
are based on the simultaneous hybridization of 24 chromosome-speci®c painting probes labelled with dierent combinations of ®ve ¯uorochromes. In combined binary ratio labelling FISH (COBRA FISH),19 the discrimination of chromosomes is based on the ratio of the hybridization signal intensities of three or four ¯uorochromes, whereas in colour-changing karyotyping, it is based on the combinatorial labelling of chromosomes.20 All these multicolour systems can detect numerical and interchromosomal aberrations in a single hybridization, but intrachromosomal aberrations, such as paracentric or pericentric inversions, small deletions and duplications, remain impossible to identify. This is resolved by the most recent re®nements of FISH, which include multicolour chromosome banding, based on the use of dierently labelled, overlapping microdissection libraries,21,22 and the simultaneous dierential painting of each chromosome arm apart from the short arms of the acrocentric chromosomes.23 Another notable enhancement is centromere-speci®c multicolour FISH (cenM-FISH), which distinguishes all the centromeric regions except those of the evolutionary highly conserved chromosomes 13 and 21.24 The increased resolution provided by dierent FISH technologies, alone or combined, has been shown in numerous studies of structural chromosome abnormalities. These include microdeletion syndromes, cryptic duplications or translocations, complex rearrangements and marker chromosomes.9±13,15±16,18±19,22,24±25 In prenatal diagnosis, dierent FISH technologies have been applied to identify and characterize structural chromosome abnormalities that cannot be resolved or diagnosed by conventional chromosome banding techniques.
Presence of a marker chromosome The identi®cation of marker chromosomes during prenatal chromosome analysis raises serious concerns regarding the phenotypic consequences to the fetus and can create dicult counselling problems.26±28 These small chromosome markers are structurally abnormal and cannot be identi®ed with conventional chromosome banding techniques. In order to avoid ambiguities, the terms `structurally abnormal chromosome' and `extra structurally abnormal chromosome' (ESAC) have been coined. The terms ESAC and `supernumerary marker chromosome' are synonymous.
634 T.-H. Bui, E. Blennow and M. NordenskjoÈld
The incidence of ESAC has been estimated to be 0.2±0.72 per 1000 newborn infants. The prevalence of marker chromosomes found in larger prenatal series also varies considerably, with a reported range of 0.6±1.5 per 1000.26 The chromosomal origin and composition of ESACs are directly associated to their phenotypic presentation. ESACs that are well-established chromosomal syndromes include isochromosome 9p, isochromosome 12p mosaicism (Pallister±Killian syndrome), isochromosome 18p and the inverted duplications inv(15) and dup(22) (cat eye syndrome). These ESACs can generally be diagnosed or suggested by conventional chromosome banding techniques. FISH is only used to con®rm the diagnosis when some doubt remains. The introduction of FISH methods in the early 1990s using chromosome-, locus- or centromere-speci®c probes has provided a powerful approach to the identi®cation of ESACs (reviewed in reference 29). Methods for de®ning ESACs are now fairly well established.27,29 Previously, each probe (or a combination of a few probes) was tried in turn to de®ne the ESAC, making the whole process highly inecient and time-consuming.30 Reverse painting using degenerate oligonucleotide primed-PCR libraries is the most informative way to outline an ESAC.31 This can be achieved by isolating the ESAC by ¯ow sorting or microdissection and using it as a template to generate an ESAC-speci®c library by PCR.31±33 Not only does this reveal the origin of the chromosome(s) involved, but it also pinpoints the precise chromosomal segments that are contained within the marker (Figure 2). When reverse painting is not available, M-FISH or SKY will, in most cases, elucidate the origin of the ESAC in a single hybridization.12,18 The cenM-FISH technique is particularly helpful for the characterization of small ESACs with no (or nearly no) euchromatin when, in addition, the amount of available sample material is restricted, as in prenatal cases.24 The most common group of ESACs involves only the paracentromeric region of a chromosome, often with satellites at one or both ends. Using FISH, about 80% of ESACs have been shown to be derived from the acrocentric chromosomes. Many of these are either pseudo idic(15), also called inverted duplication, inv dup(15), or ESACs derived from chromosome 22.26,34 When the inv dup(15) includes the Prader±Willi/ Angelman critical region, it is associated with mild-to-severe mental retardation and other pathological features.35,36 Chromosome 22 ESACs without euchromatin are not associated with an adverse phenotype. Rare analphoid marker chromosomes with a neocentromere have been reported (reviewed in reference 37). About 40% of ESACs found on prenatal analysis are inherited, and the risk of an adverse phenotype in the fetus is usually low if the carrier parent has a normal clinical phenotype. The residual risk results from the possibility of uniparental disomy38, i.e. the inheritance of both homologous chromosomes from only one parent, or the fact that the fetus has inherited an ESAC in a non-mosaic form from a mosaic parent. A review of de novo cases of non-acrocentric, autosomal ESACs characterized by FISH gave a risk estimate of an abnormal phenotype of about 28%.27,28 ESACs derived from acrocentric chromosome may result in a risk comparable to that of the other autosomes if de novo supernumerary marker chromosomes derived from chromosomes 15 and 22 ± of which the majority are clinically normal ± are excluded. The unequivocal identi®cation of the origin and composition of ESACs is clearly crucial for karyotype±phenotype correlation and a more accurate assessment of risk in individual cases. Ultrasound examination for fetal anomalies may also assist in this individual assessment.27
Prenatal diagnosis: molecular genetics and cytogenetics 635
Figure 2. (A) Fluorescence in situ hybridization (FISH) on a metaphase chromosome spread with a chromosome 8-speci®c painting probe, showing the two normal chromosomes 8 (red colour) and the origin of a small ring marker. (B) Microdissection of the supernumerary ring marker chromosome that will be used as a template to generate a marker-speci®c library by a polymerase chain reaction. (C) FISH using the marker-speci®c probe reveals not only the origin of the ring marker chromosome, but also the chromosomal segments involved and the ®nal karyotype: 47,XX,r(8)(q10q21.1)de novo.
RAPID PRENATAL DIAGNOSIS OF THE MOST COMMON NUMERICAL CHROMOSOME ABNORMALITIES Two methods have recently been introduced for the rapid prenatal diagnosis of numerical chromosome abnormalities. These are interphase FISH and quantitative ¯uorescent polymerase chain reaction (QF-PCR) assay. Interphase FISH The most widespread application of FISH in prenatal diagnosis is for the rapid detection (1±2 working days) of the common numerical chromosome abnormalities using chromosome-speci®c probes applied to interphase cells from amniocenteses and chorionic villi samples (Figure 3), thus obviating the 7±14 days' delay resulting from cell culture associated with conventional chromosome analysis.39±42 Most commonly,
Figure 3. Interphase ¯uorescence in situ hybridization on uncultured amniocytes. (A) Three green signals for a chromosome 13-speci®c probe (there being in one nucleus a partial masking of one red signal by one green signal). Thus, the fetus carries a trisomy 13. (B) Three signals for each of the chromosome-speci®c probes used in three nuclei from the same amniotic ¯uid sample. Left, three green signals for a chromosome 13 probe; middle, three red signals each for chromosome 13 probe and three signals for the chromosome 21 probe; right, three light blue signals for the chromosome 18 probe. The fetus therefore has a triploidy.
636 T.-H. Bui, E. Blennow and M. NordenskjoÈld
Prenatal diagnosis: molecular genetics and cytogenetics 637
probes speci®c for chromosomes 13, 18, 21, X and Y are used as, depending on the indications for invasive tests, numerical abnormalities involving these chromosomes account for 70±95% of the chromosome aberrations identi®ed prenatally.9±11,39±43 The usefulness of interphase FISH in prenatal diagnosis and the test performance characteristics have been recently reviewed in 29 039 informative testing events using one commercially available assay (AneuVision, Vysis Inc, Downers Grove, IL, USA) and 18 275 other specimens tested with a diversity of other probes.39 With the AneuVision assay, the detection rate was nearly 100%, with a false-positive rate of only 0.003% and a false-negative rate of 0.024%. In many centres with a wide experience of interphase FISH, results are now acted upon if, for example, a common trisomy is found, without waiting for the examination of a complete karyotype after cytoculture. The diagnostic capability of interphase FISH is obviously limited by the choice of probe used: structural chromosome aberrations and numerical chromosome abnormalities other than those tested for will be missed. In addition, the incomplete hybridization of FISH probes or a reduction in signal size due to heteromorphisms39,44±47 may lead to falsenegative prenatal FISH results. More recent FISH probe sets use locus-speci®c probes that avoid the problems of centromeric heteromorphisms. In cases of ambiguous FISH results, however, full karyotyping should remain the gold standard. Recently, chromosome arm-speci®c subtelomere probes have been used successfully to rapidly determine the segregation of a parental balanced reciprocal translocation in uncultured amniocytes,48,49 an approach similar to that used in pre-implantation genetic diagnosis on single cells (see Chapter 4 in this issue). Chromosome arm-speci®c pro-telomere probes are available for each chromosome,13 and it would be possible to oer a rapid diagnosis for similar indications in selected couples.
Quantitative ¯uorescent polymerase chain reaction An alternative approach to interphase FISH is to employ a QF-PCR assay, which also allows the detection of major numerical chromosome disorders within a few hours of sample collection. Highly polymorphic chromosome-speci®c STRs are ampli®ed by a PCR. A ¯uorochrome incorporated into the products of ampli®cation permits visualization and quanti®cation using an automated DNA scanner. Two peaks of ¯uorescence activity with a ratio of about 1:1 will be produced for each chromosomespeci®c STR from samples obtained from normal heterozygous fetuses. 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 (trisomic diallelic). The principle of the QF-PCR test using STRs is illustrated in Figure 4.50 If three or more polymorphic STR markers are used for each chromosome analysed, very few fetal samples will remain uninformative as a result of homozygosity at one locus. In recent years, QF-PCR has been successfully applied to determine the copy number of ®rst chromosomes 13, 18, 21 (Figure 5) and later chromosomes X and Y in uncultured amniocytes or chorionic villi for prenatal diagnosis (reviewed in reference 50).51±57 The advantage of QF-PCR over FISH is that it is considered substantially more cost-eective, especially when larger sample numbers are processed: less material is required, bloody specimens may be successfully processed, and QF-PCR is less time-consuming and labour intensive. However, the same limitations concerning the overall detection rate of unbalanced chromosome anomalies apply for both techniques as currently applied.
638 T.-H. Bui, E. Blennow and M. NordenskjoÈld
Figure 4. Principle underlying the quantitative ¯uorescence polymerase chain reaction for the rapid prenatal diagnosis of common aneuploidies. Reproduced with permission from Adinol® et al (2000).50
FUTURE PROSPECTS AND CONCLUSIONS Over the past decade, an impressive array of technologies has emerged to analyse variant DNA and structure in the human genome. Many of these methods have now been incorporated into the arsenal available to routine molecular genetics laboratories. DNAbased prenatal diagnosis is currently targeted towards families at risk of an inherited condition and is not used as a screening test because of cost, technical complexity and limited resources. In the future, an increasing number of single-gene disorders will be amenable to testing from chorion villus cells or amniocytes and also potentially from fetal cells isolated from the maternal circulation or fetal DNA in maternal plasma.58±59 New analytical devices including comparative genome hybridization11 and microchips60
Prenatal diagnosis: molecular genetics and cytogenetics 639
Figure 5. Partial electrophoretograms of the quantitative ¯uorescent polymerase chain reaction products of dierent chromosome 21-speci®c short tandem repeat markers. (A) Normal disomic heterozygote (ratio 1:1). (B) Trisomic triallelic (ratio 1:1:1) for marker D21S226 and trisomic diallelic (ratio 2:1) for marker IFNAR. The fetus thus has trisomy 21. (C) Trisomic triallelic (ratio 1:1:1), indicating a fetus with trisomy 21. Reproduced with permission from Dr HeleÂne Olsson, CyberGene AB, Huddinge, Sweden.
640 T.-H. Bui, E. Blennow and M. NordenskjoÈld
are being applied to molecular genetics and are likely to ®nd applications in clinical medicine. Eorts to develop better methods for target probe and signal ampli®cation will result in better DNA detection tools.61 The introduction of multicolour and other FISH technologies has contributed signi®cantly to the detailed characterization of cryptic or subtle structural chromosome abnormalities, complex rearrangements and ESACs in prenatal diagnosis, as well as providing a better understanding of their clinical signi®cance. Both interphase FISH and QF-PCR are ecient tools for the rapid prenatal diagnosis of common aneuploidies, although current experience suggests that QF-PCR is more cost-eective in large-scale application. Whether QF-PCR should be performed as an adjunct to full karyotyping or be used as a stand-alone test, at least for some select indications, is currently under debate. Acknowledgement Supported in part by the European Commission (ENFET concerted action).
Practice points . an extensive genetic work-up and counselling are needed before DNA-based prenatal diagnosis can be oered for selected single-gene disorders . karyotyping remains the `gold standard' in prenatal diagnosis . dierent FISH technologies provide increased resolution and allow the de®nition of structural chromosome abnormalities that cannot be resolved by conventional chromosome analysis . both interphase FISH and QF-PCR allow the rapid (1±2 day) prenatal diagnosis of common aneuploidies . QF-PCR appears to be more cost-eective for large-scale use
Research agenda increased pace in the discovery and characterization of disease-causing genes further development in technologies for mutation analysis and FISH further studies on fetal DNA and cells in the maternal circulation further large-scale clinical studies of QF-PCR for the rapid prenatal diagnosis of selected numerical abnormalities . better methods for the ampli®cation of target, probes and signals for DNA detection tools . . . .
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