Prenatal Diagnosis

Victoria Binns, Swedish Medical Center, Seattle, Washington, USA Nancy Hsu, Swedish Medical Center, Seattle, Washington, USA
Prenatal diagnosis is the process of ruling in or out fetal anomalies or genetic disorders, to provide expecting parents with information and the opportunity to modify pregnancy management and/or postnatal care.

Secondary article
Article Contents
. Introduction . Indications for Prenatal Diagnosis . Methods of Prenatal Diagnosis . Chromosome Analysis . Karyotype Analysis . Fluorescence in situ Hybridization (FISH)

Introduction
Researchers are gaining knowledge about the genetic basis of heritable disorders, allowing medical professionals to be increasingly equipped to diagnose such disorders in utero. Although some genetic disorders are compatible with long, healthy lifespans, many are associated with signicant morbidity, mortality and mental retardation. Expectant parents have many options available for prenatal screening and testing for genetic disease. By identifying genetic disorders in utero, parents and professionals can make decisions regarding pregnancy maintenance and management. Yet prenatal diagnosis opens the door to a whole new era of medicine, where the ability to diagnose genetic disease often precedes the ability to treat or cure. Ethical principles are intertwined with prenatal genetic testing; those seeking and providing it often face controversial decisions and ethical dilemmas. Each case requires an integrated team approach involving the patient, laboratory, genetic professionals and other specialists to ensure maximum options and most appropriate care.

. DNA Molecular Analysis . Linkage Analysis . Biochemical Analysis . New Diagnostic Techniques . Genetic Counselling . Ethics

Indications for Prenatal Diagnosis

Advanced maternal age
One of the most common indications for prenatal diagnosis is advancing maternal age (Table 1); as a womans age increases, so too does the risk for chromosome aneuploidy in the fetus (Figure 1). While aneuploid conceptions are not uncommon, the majority are misTable 1 Indications for prenatal diagnosis . . . . . . . Advanced maternal age Multiple pregnancy losses (3) Known or suspected family history of genetic disease or multifactorial disorder Ethnicity at increased risk for genetic disease Teratogen Abnormal ultrasound ndings Abnormal maternal serum screen results

carried during the rst trimester of pregnancy (Gardner and Sutherland, 1996). Fifty per cent of chromosomally abnormal fetuses are trisomic, with trisomy 16 being the most frequent. Aneuploidy is the result of meiotic nondisjunction, where the failure of homologous chromosomes to separate during anaphase results in one gamete containing both homologues, the other containing none (Figure 2). Upon fertilization, the conceptus is either monosomic or trisomic for the given chromosome (provided the partners gamete is chromosomally normal). Evidence suggests that lack of recombination between homologues predisposes chromosome pairs to move to the same pole during meiosis I (Sherman et al., 1994); however, related research is ongoing. Ninety per cent of nondisjunction events are of maternal origin, three-fourths arising in anaphase I (Gardner and Sutherland, 1996). The remaining 10%
15 14 13 12 11 10 9 8 7 6 5 4 3 2 1 0 24

% Risk

28

32

36 Maternal age

40

44

48

Figure 1 Risk of chromosomal abnormalities by maternal age. Adapted from Ferguson-Smith and Yates (1984), Hook and Chamber (1977), Hook (1981 and 1990).

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Prenatal Diagnosis

46

46

23

23

23

23

23

23

23

23

24

22

23

23

47
Figure 2 Normal meiosis (left) and nondisjunction (right). Only one of the 23 chromosome pairs is demonstrated; assume all other chromosome pairs undergo normal meiosis. Numbers reflect the total number of chromosomes in the gamete at that stage in meiosis.

occur due to nondisjunction during either meiotic division or spermatid development (Abruzzo and Hassold, 1995). In theory, nondisjunction may involve any of the 23 pairs of chromosomes, although the majority are believed to result in early miscarriage. Aneuploidies that may survive to term include trisomies 13, 18 and 21, and monosomies and polysomies involving the sex chromosomes. Clinical manifestations are dependent upon the chromosome involved. Trisomy 21 results in classic Down syndrome, with variable degrees of mental retardation and increased risk of heart and intestinal defects (Figure 3). Trisomies 13 and 18 are associated with signicantly increased perinatal mortality due to multiple congenital anomalies, whereas sex chromosome aneuploidies (XXY, XYY, XXX) have highly variable prognoses, aecting fertility, physical appearance and mental status (Gardner and Sutherland, 1996). Although rare, complete nondisjunction of all chromosome pairs in either the egg (diandry) or sperm (digyny) may also occur, and result in a triploid (3n 5 69 chromosomes) conceptus, which is associated with high prenatal mortality. Amniocentesis and chorionic villus sampling (CVS) are techniques for obtaining fetal karyotype. Although increased risk of numerical chromosome abnormalities due to increasing maternal age is the foremost indication for prenatal diagnosis, resultant karyotypes may also reveal structural chromosome abnormalities, such as translocations, inversions, insertions, deletions or rings.
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Information regarding the natural history and prognosis of particular aneuploidies and chromosomal rearrangements should be given to parents after prenatal diagnosis. Concerns about raising a child with special needs and the possible option of termination should be explored in a supportive, sensitive manner. Regardless of the decision to continue or terminate a pregnancy, parents should be reassured that recurrence risk for aneuploidy is not signicantly increased following the birth of an aected child. For women under 35, the recurrence risk is  1% (Gardner and Sutherland, 1996). For women over 35, the recurrence risk resets to the mothers maternal age-related risk.

Multiple miscarriages and/or fetal losses

Causes for multiple miscarriages (three or more) can be chromosomal, anatomical, immunological or hormonal. Approximately half of recognized miscarriages are due to chromosome abnormalities (Gardner and Sutherland, 1996); of these, 2% have unbalanced translocations. To rule out inherited translocation, parental blood may be drawn for karyotype analysis. Balanced translocation carriers, and couples who have had three or more miscarriages, may be interested in fetal chromosome analysis via amniocentesis or CVS during future pregnancies. Couples with a history of unexplained loss may,

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Prenatal Diagnosis

Figure 3 (a) Normal karyotype. (b) Karyotype of an individual with Down syndrome, characterized by an extra chromosome 21, provided by either egg or sperm. This karyotype is written: 47,XY, 1 21, demonstrating that there are 47 total chromosomes, the sex chromosomes are X and Y (male), and the extra chromosome is a number 21. For a female with Down syndrome, the karyotype would be: 47,XX, 1 21. Courtesy of Dynagene Cytogenetics Laboratory, Swedish Medical Centre, Seattle, WA, USA.

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Prenatal Diagnosis

however, be especially sensitive to the risks of these techniques. If this rearrangement does not result in the addition or loss of genetic information, the karyotype is described as balanced: the individual is phenotypically unaected. If the rearrangement does cause genetic information to be lost and/or gained, the karyotype is described as unbalanced: the individual is phenotypically aected (displays signs and/or symptoms). Parents with balanced chromosome arrangements are at risk of having children with unbalanced rearrangements.

chromosomal or genetic disease, but may lead to fetal abnormality, distress or demise. Prenatally, abnormal ultrasound ndings may suggest maternal exposure; if this is suspected or identied during pregnancy, specic management may be oered throughout. Information required to investigate the specic teratogenic eects of a given exposure includes the following: . Type of agent. This can determine the likelihood that a potential teratogen will enter the bloodstream, cross the placenta and aect the fetus, or be transmitted in high concentration in breast milk. . Timing and duration of exposure. Teratogens often have the greatest adverse eect during a specic period of embryonic development. . Dosage. Teratogens often only cause detrimental eects when present above a threshold amount. (Unfortunately, a given substances threshold is often not clear.) . Route of administration. Ingestion may allow a higher concentration to enter the maternal bloodstream than other modes of administration (such as inhalation or topical application.) Determining a substances eect upon a fetus is dicult, as the majority of human clinical studies available are retrospective and subject to recall bias. Although there are known teratogens, many exposures are associated with little or no teratogenic risk. Eliminating exposure, or limiting it to the lowest eective dosage is ideal; however, it is important to weigh maternal well-being against the potential for fetal teratogenesis. (For example, if a pregnant woman is at risk of grand mal seizures when not medicated, the benet gained from medicating, and avoiding risk to mother and fetus due to seizures, may outweigh the potential risk of congenital anomalies associated with a particular seizure medication.)

Known or suspected family history of genetic disease or multifactorial disorder

If a monogenic, polygenic or multifactorial disorder is in the family history, the pregnancy may be aected: analysis of family history is often used to assess the suspected disorders inheritance pattern and thus accurately assess risk to the fetus. Conrmed family history, accurate diagnosis and reliable testing are key factors in prenatal diagnosis.

Ethnicity at increased risk for genetic disease

Certain ethnic groups have an increased incidence of specic genetic conditions due to lack of migration, genetic drift or heterozygote advantage within the given population or geographic area. If these inherited mutations cause disease in the homozygous state, members of the given population are at increased risk of having children aected with recessive conditions. Population screening has been developed to screen members of at-risk populations for particular gene mutations: yet sensitivity and specicity are directly related to the prevalence of the mutation within that population. Thus, some populations at increased risk have more accurate screening available, as their increased disease prevalence is due to a handful of mutations that have become readily identiable. (patients should be informed of the sensitivity and specicity of carrier screening and prenatal diagnosis before proceeding). For example, Tay Sachs disease is more common in the Ashkenazi Jewish population. Three mutations account for 94% of disease carrying mutations in the Ashkenazi population. This type of DNA mutation analysis is less accurate for those of nonAshkenazi background; therefore enzyme analysis is more accurate and appropriate.

Abnormal ultrasound findings

Abnormal ultrasound ndings may prompt invasive prenatal diagnostic measures (amniocentesis or CVS) if fetal structural abnormalities or markers associated with chromosome conditions are identied. For example, ultrasound may suggest the presence of chromosomal aneuploidies such as Down syndrome, genetic syndromes such as dwarsm, hereditary renal disorders or isolated birth defects.

Teratogens
Maternal disease (e.g. insulin-dependent diabetes, maternal phenylketonuria), infection (e.g. toxoplasmosis, rubella) or exposure to internal or external substances (e.g. medications, alcohol, radiation) are not associated with
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Abnormal maternal serum screen results

Maternal serum screening may identify women at increased risk of having a child with Down syndrome, trisomy 18 or an open neural tube defect (spina bida). In these cases, positive serum screen results often prompt consideration of diagnostic testing by amniocentesis.

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Prenatal Diagnosis

Methods of Prenatal Diagnosis

Chorionic villus sampling
CVS was rst used for prenatal diagnosis in the 1970s and 1980s. It can be used to yield information on fetal chromosome status, diagnose single gene disorders (via uorescence in situ hybridization (FISH)) or assay for biochemical disease. It is typically performed at 1012 menstrual weeks of pregnancy; when chorionic villi are successfully obtained, karyotype results are 499% accurate, and therefore considered diagnostic. CVS provides insight about embryonic karyotype, as both placenta and embryo are derived from the same totipotent stem cell (initial undierentiated cell), before it further develops and forms several cell lineages (Figure 4). In its initial stages, this cell begins dividing, rst becoming a morula (ball) of 1215 undierentiated cells, from which some develop into chorionic ectoderm (trophoblast) and some into chorionic mesoderm. Both of these are sampled via CVS direct and long-term culture. Still other cells remain, which eventually dierentiate to become amniotic mesoderm, ectoderm and endoderm, to be sampled at the time of amniocentesis or percutaneous umbilical blood sampling (PUBS) (Moore and Persaud, 1998). Procedure During CVS, samples of trophoblast cells and chorionic villi are withdrawn transabdominally or transcervically, depending upon placental and uterine position, physician

recommendation and/or patient preference (Figure 5). In the laboratory, extraneous maternal cells (decidua) are separated from the fetal specimen to produce a sample that will reect the fetal karyotype. Ideally, 1020 mg of villi are withdrawn with each sample, using a needle or catheter lled with tissue culture medium. The turnaround time for CVS results depends upon which cell line is analysed. Trophoblast cells (the rst to dierentiate from the morula) may be analysed directly (same day) or after short-term culture (12 days). The villus core, containing the capillaries, is used for long-term culture, as it comprises further-dierentiated cells (representing the extraembryonic mesoderm). Long-term culture results, available after approximately 2 weeks, are often used to conrm those of short-term culture. Early studies correlated CVS with fetal limb reduction defects (Firth et al., 1991). Numerous subsequent investigations have failed to completely conrm the association. Although still controversial, it is generally accepted that between 10 and 12 weeks, there is no increase in limb anomalies over the background risk. Outside this timeframe, the risk may be increased (see review by Burton et al., 1992).

Amniocentesis
Amniocentesis was rst performed in 1952 to diagnose haemolytic disease prenatally. By the mid-1970s, it became a standard for obtaining fetal karyotype. Currently it is

d Trophoblast (chorionic ectoderm) CVS direct preparation

Undifferentiated cell Extraembryonic ectoderm

Extraembryonic mesoderm Embryonic ectoderm or endoderm Embryonic mesoderm

b 1

b 2

c c
ys hy ys

Chorionic mesoderm ? Fetal blood Amniotic mesoderm Amniotic ectoderm Ectoderm Mesoderm ? Fetal blood Ectoderm

CVS culture

3
ep

c b

Amnio centesis Fetal biopsy

ps

Figure 4 Development of various cell lines in the human embryo. The fertilized egg (1) gives rise to a trophoblast precursor (1b) and a totipotent stem cell (2), which produces another trophoblast precursor (2b) and a stem cell (3), which give rise to the inner cell mass. This divides into stem cells and becomes the hypoblast (hy, 3b) and epiblast (ep, 4). Only a few of the epiblast cells go on to form the embryo in the inner cell mass. (5) CVS, chorionic villus sampling. ys, yolk sac; ps, primitive streak. Reproduced with permission of Wiley Liss Inc., New York, from Bianchi DW et al. (1993) Origin of extraembryonic mesoderm in experimental animals: relevance to chorionic mosaicism in humans. American Journal of Medical Genetics 46: 542 550.

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Prenatal Diagnosis

Ultrasound probe

Chorionic villi Uterine wall Placenta Amniotic fluid Chorionic villi Needle

(a)

Ultrasound probe Amniotic fluid Chorionic villi Uterine wall Placenta Biopsy catheter Cervix

(b)
Figure 5 (a) Transabdominal and (b) transcervical chorionic villus sampling. Courtesy of Dr Robert Saul, Greenwood Genetic Center, Greenwood, SC; from Counseling Aids for Geneticists, 3rd edn, 1995.

typically performed after 15 weeks gestation, although it may be performed earlier if circumstances dictate. Amniotic uid contains exfoliated fetal cells that may be cultured to reveal fetal karyotype, and/or perform biochemical testing and molecular analysis. Additionally, because amniotic uid is removed along with suspended fetal cells, levels of alpha-fetoprotein (AFP) are commonly measured to screen for neural tube and abdominal wall defects. Elevated levels of AFP in the amniotic uid suggest the
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presence of such birth defects; if identied, additional screening by fetal ultrasound and for the presence of acetylcholinesterase (AChE) in amniotic uid may clarify whether an open neural tube or ventral wall defect is present. Procedure Under ultrasound guidance, a needle is inserted through the mothers abdomen and uterus into the amniotic sac,

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Prenatal Diagnosis

Ultrasound probe

Amniotic fluid Placenta Fluid -Fetoprotein Acetylcholinesterase Centrifuge Cells Biochemical studies DNA studies

Cell culture Chromosome analyses DNA studies Biochemical studies

Figure 6 Prenatal diagnosis by amniocentesis. Courtesy of Dr Robert Saul of Greenwood Genetic Center, Greenwood, SC; from Counselling Aids for Geneticists, 3rd edn, 1995.

Table 2 Amniocentesis or chorionic villus sampling (CVS)? . . Risk of complications. The risk of complications and/or miscarriage in amniocentesis is about 1/200; whereas in CVS the risk is about 1/100. Parents may opt for amniocentesis over CVS because of the lower risk of amniocentesis Gestational age. CVS provides information about the pregnancy at an earlier gestational age, allowing more time for parental adjustment and/or decision-making. For some, termination of pregnancy is less dicult, both emotionally and physically, during the earlier stages of pregnancy Accuracy of test results. Although both techniques are considered diagnostic, there is a higher likelihood of ambiguous results in CVS because approximately 12 % of CVS samples reect conned placental mosaicism, wherein the placenta, but not the fetus, contains both normal and abnormal cell lines. There is also a possibility of maternal cell contamination, wherein maternal cells are not completely distinguishable or separated from fetal cells, and may be included in cell analysis. In such cases, follow-up ultrasound, parental karyotyping and/or amniocentesis may be oered Extent of test. Because CVS cannot detect the risk of a neural tube defect or abdominal wall defect, follow-up screening of maternal blood by a-fetoprotein serum screening is recommended at 15 weeks gestation

from which approximately 20 mL of amniotic uid are withdrawn (Figure 6). Fetal cells are isolated from the uid and cultured for analysis. While karyotype results are typically available in 1014 days, the time required for deoxyribonucleic acid (DNA) and biochemical results varies, depending upon the assay required. Because CVS and amniocentesis ultimately provide the same information about a fetus, it is important that

patients recognize the dierences between the procedures (Table 2).

Percutaneous umbilical blood sampling

Typically performed after 18 gestational weeks, PUBS (cordocentesis) is a method of obtaining fetal blood from
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Prenatal Diagnosis

Fetal blood Fetal blood Ultrasound probe

Placenta Umbilical cord

Umbilical vein Umbilical arteries

Uterine wall

Figure 7 Percutaneous umbilical blood sampling. Courtesy of Dr Robert Saul of Greenwood Genetic Center, Greenwood, SC; from Counseling Aids for Geneticists, 3rd edn, 1995.

the umbilical vein under ultrasound guidance (Figure 7). A needle is inserted through the mothers abdomen to obtain a fetal blood sample. The procedure-related fetal loss rate is estimated at 12% (Tongsong et al., 2000). PUBS may be used to diagnose fetal toxoplasmosis and haematological disorders, by measuring increases or decreases of particular blood factors. It can also be used to diagnose chromosome instability syndromes (e.g. Fanconi anaemia), prenatal infection and aneuploidy, and at times is used for in utero blood transfusions. As the molecular biology of genetic disorders continues to unfold and DNA techniques improve, PUBS is beginning to be replaced by direct DNA analysis of CVS or amniocentesis samples.

unaected blastocyst is implanted into the uterus. The small possibility of incorrect results associated with meiotic crossing-over between sister chromatids is avoided (unlike linkage analysis, described below), although mosaicism of blastocyst cells may still occur and lead to misdiagnosis. An advantage to preconception testing over traditional postconception prenatal diagnosis is that it allows parents to avoid the possibility of receiving abnormal prenatal diagnosis results, and thus the dicult decisions associated with pregnancy management and/or maintenance. PGD can be laborious, time-consuming and expensive. Complicating factors include a high rate of polyspermia, a small amount of DNA in polar bodies (making it dicult to amplify) and meiotic crossing-over, which can produce less denitive test results.

Preimplantation genetic diagnosis and preconception testing

Preimplantation genetic diagnosis (PGD) has been practised for approximately a decade; the most frequent candidates are parents with family histories of serious monogenic disorders and translocations, who are therefore at increased risk for transmitting these conditions to future generations. Polar body and blastomere testing are the two primary methods of PGD. In polar body testing, positive test results in two polar bodies ensure that the egg itself is unaected therefore, the mutation has segregated to the polar body, not to the developing ovum. Once an egg is found to be unaected, it is fertilized via traditional in vitro fertilization (IVF) and implanted into the uterus. Blastomere PGD rst requires traditional in vitro fertilization, after which cells are grown to the 8-cell stage. One or two cells are harvested and analysed, and an
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Abnormal ultrasound findings

Ultrasound is used in the second trimester to identify major fetal structural anomalies and fetal anatomical markers, either of which may be associated with underlying chromosomal disorders (e.g. Down syndrome), single gene disorders (e.g. achondroplasia) or normal variation. An anatomical marker is a sonographic nding that occurs more commonly in aected than unaected fetuses. Its presence raises the possibility of a particular disorder but does not itself identify the fetus as having the disorder. For example, a fetal echogenic bowel may signify a chromosome abnormality, cystic brosis (CF), intraamniotic haemorrhage, fetal infection, or normal variation. Follow-up testing (e.g. amniocentesis) may be oered to rule out or diagnose when abnormal ultrasound suggests an underlying genetic disorder, and/or when family history

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Prenatal Diagnosis

Figure 8 Ultrasound image demonstrating the measurement of nuchal translucency (the nuchal fold) at the back of the fetal neck. Increased nuchal thickness (42 mm in the first trimester; 45 mm in the second trimester) has been associated with increased risk of Down syndrome (and other aneuploidies) and is therefore considered a marker for these conditions. Courtesy of Dr Vivienne Souter, Swedish Medical Center, Seattle, WA, USA.

Table 3 Screening (ultrasound) versus diagnostic testing (amniocentesis, chorionic villus sampling) Screening Risk of complications or miscarriage Information provided Timing of results None Limited Instantaneous Diagnostic testing Exists; dependent upon technique: Amniocentesis: about 1/200 Chorionic villus sampling about 1/100 Diagnostic (499% accuracy if gene mutation known) Results available in days to weeks

suggests an increased risk. Approximately 50% of fetuses with Down syndrome can be identied by ultrasound by detection of markers such as increased nuchal translucency, shortened femoral and/or humeral length, identication of an echogenic cardiac focus, hyperechogenic bowel, and/or dilation of cerebral ventricles (Figure 8). Ultrasound screening is not diagnostic; moreover, abnormal ndings may be transient, aected fetuses may not have detectable anomalies, and unaected fetuses may show sonographic markers, simply as a matter of normal variation. Ultrasound may identify the presence of a specic diagnosis, such as spina bida, anencephaly or dwarsm, but may not be able to diagnose the severity of the spina bida or the specic form of dwarsm. When such an anomaly is diagnosed on ultrasound, pedigree and DNA analysis may be able to pinpoint the specic diagnosis.

It is important for parents to understand the dierences between screening and diagnostic testing (Table 3). Screening measures such as ultrasound pose no risk to the pregnancy but ndings are based upon views of the fetus, the estimated gestational age, sonographer experience, and the degree of anomaly severity. Diagnostic testing (CVS and amniocentesis) results are considered conclusive, although the invasive nature of the procedures poses a risk for complications and miscarriage.

Maternal serum screening

Maternal serum screening is used to identify women at increased risk of having a child with trisomies 18 or 21 or an open neural tube defect (NTD), while posing no risk to the pregnancy. Levels of AFP, human chorionic
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Prenatal Diagnosis

gonadotrophin (HCG) and unconjugated oestriol (UE3) are measured between 15 and 18 weeks gestation. These substances are of fetal origin and cross from the amniotic uid into maternal circulation via the placenta. Substance levels, maternal age, weight, race, diabetic status, pregnancy history and gestational age are taken into account to rene maternal age-related risks. Low maternal serum AFP, low UE3 and/or elevated HCG levels are associated with increased risks of fetal Down syndrome, whereas low levels of all three substances suggests increased risks for trisomy 18 or triploidy. High levels of AFP are associated with increased risk of neural tube and abdominal wall defects; while high levels of HCG can be associated with increased risk for pregnancy complications. Maternal serum screening detects approximately 60% of Down syndrome, although the sensitivity and false-positive rate vary with the age of the mother. Although the exact association between abnormal ultrasound ndings and maternal serum screening is unclear, both techniques can provide couples with more information regarding their pregnancies, and oer the possibility of further diagnostic prenatal testing. Recent advances in maternal serum screening involve incorporating a fourth substance, inhibin A, to analysis, and performing screening during the rst trimester, in conjunction with sonographic measurements of nuchal translucency. Both these measures are expected to increase the sensitivity of maternal serum screening, while keeping false-positive results at a minimum.

banding is typically used rst to analyse prenatal specimens, various other banding techniques (including quinacrine (Q), reverse (R), centromeric heterochromatin (C) and high-resolution banding) may be used to analyse dierent portions of particular chromosomes (heterochromatin, nucleolar organizer regions, etc.).

Fluorescence in situ Hybridization (FISH)

FISH is mainly used to detect the presence or absence of microdeletions, microduplications and aneuploidy without the full eort associated with DNA sequencing or complete karyotype analysis. This three-step technique allows specic DNA sequences or chromosomes to be visualized microscopically. A specic, single-stranded DNA probe is hybridized to its complementary, target DNA sequence, while the cell is in prophase, metaphase or interphase; uorescent antibodies are then hybridized to the probe DNA sequence; nally, the uorescent signals are examined under the microscope. FISH analysis for common aneuploidies (involving chromosomes 13, 18, 21, X and Y) is often performed by simultaneously applying specic multicoloured centromeric probes. In fetal trisomies, three probes are present for a specic chromosome, while monosomies show only one. Additionally, dierent coloured probes may be used in combination in order to analyse multiple chromosomes, while unique sequence probes, specic for a predicted submicroscopic deletion or duplication, can be used to detect the presence of specic monogenic disorders (Figure 9, Tables 4 and 5).

Chromosome Analysis
Chromosome analysis is a technique used to identify aneuploidy, microdeletions, microduplications and major structural aberrations. The most common method of detecting aneuploidy is karyotype analysis, wherein metaphase cells are examined microscopically and the number of chromosomes counted. Typically 1015 cells are analysed to rule aneuploidy in or out; however, a larger number of cells may be analysed to investigate the presence of mosaicism.

Karyotype Analysis
Each chromosome pair has a unique banding pattern that can be seen with various stains. The most common method of karyotype analysis is Giemsa (G) banding, wherein chromosomes are denatured (with trypsin), revealing a pattern of light and dark bands. Counting the number of staining chromosomes allows for detection of aneuploidies. Analysing for the absence, presence, rearrangement, etc. of these bands allows for detection of larger deletions, duplications and structural aberrations. Although G
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Figure 9 An example of fluorescence in situ hybridization (FISH) analysis, wherein interphase nuclei from an amniocentesis sample are hybridized with probes for chromosomes 13, 18, 21, X and Y. (a) A nucleus has been hybridized with probes for chromosomes 18 (aqua), X (green) and one Y (red). (If this had been a female fetus, there would have been two green lights and no red.) (b) A nucleus has been hybridized with probes for chromosomes 13 (green) and 21 (red). There are two number 13 chromosomes (normal) and three copies of chromosome number 21 (indicating Down syndrome). Overall result: male fetus with Down syndrome. Courtesy of Dynagene Cytogenetics Laboratory, Swedish Medical Center, Seattle, WA, USA.

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Prenatal Diagnosis

Table 4 Microdeletions/microduplications detectable by fluorescence in situ hybridization (FISH) Disorder Angelman syndrome BeckwithWiedemann syndrome CharcotMarieTooth disease Choroideraemia Cri du chat syndrome DiGeorge syndrome Duchenne muscular dystrophy Grieg cephalopolysyndactyly Kalmann syndrome/ichthyosis MillerDieker syndrome PraderWilli syndrome Retinoblastoma RubinsteinTaybi syndrome SmithMagenis syndrome Shprintzen syndrome/VCFS -Thalassaemia WAGR syndrome Williams syndrome WolfHirschhorn syndrome Chromosome band 15q12 (maternal) 11p15 17p11.2 Xq21.1 5p 16 22q12 Xp21.2 7p13 Xp22.3 17p13 15q12 (paternal) 13q14.11 16p13.3 17p11.2 22q11.2 16p13.3 11q13 7q11.23 4p16 Findinga Del Dup Dup Del Del Del Del Del Del Del Del Del Del Del Del Del Del Del Del

WAGR, Wilms tumour, aniridia, genitourinary abnormalities and mental retardation. a Del, microdeletion; Dup, microduplication; VCSF, Velo-Cardio-Facial syndrome.

Table 5 Fluorescence in situ hybridization (FISH) Probes and functions Type of FISH Probe Centromeric probe Unique sequence probe Whole chromosome paints Reverse paints Comparative genomic hybridization Identies The most common aneuploidies (trisomies 13, 18, 21, X and Y) Microdeletions, microduplications Translocations Markers, supernumerary chromosomes Regions of DNA loss or amplication

FISH analysis is advantageous because results are highly reliable and typically available within a couple days after sampling. It allows expedient analysis of fetal genotype when a particular disorder is suspected and/or when parents need to make rapid decisions (due to late gestational age) about maintaining or voluntarily terminating a pregnancy. However, FISH analysis for monogenic disorders is limited because the sequence of the specic genetic mutation must be known in order to apply the correct probe; and even when such a sequence is known, FISH may not identify a disorder if it is present in only a portion of the samples cells (a mosaic sample). Finally,

FISH may be an expensive and out-of-pocket endeavour for parents.

DNA Molecular Analysis

Once a specimen is collected, a variety of laboratory techniques may be used to diagnose single gene disorders prenatally. Choosing which technique to use depends upon the suspected disorder (e.g. is enzymatic assay or mutational analysis appropriate?), how much is known about
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Prenatal Diagnosis

the disorders molecular genetics and whether other family members are aected.

HbA: normal haemoglobin gene MstII Exon I HbA: 1.1 kb HbS: sickle cell haemoglobin MstII Exon I

MstII

MstII

Direct DNA analysis

Direct mutation analysis involves analysing a target segment of DNA for the presence of a specic mutation. Like FISH, it requires knowledge of the correct sequence for the specic gene or DNA segment before analysis. Once known, the sample sequence may be compared to the known, model, genomic sequence in a variety of methods, as described below. Mutation analysis with restriction enzymes If the putative mutation is known to alter the recognition for a splice site, direct analysis by restriction enzyme assay is possible. The presence of a mutation can be detected by digesting control and sample DNA with the same restriction enzymes (known to cut the DNA at a specic splice site) and then analysing resultant DNA fragments (called restriction fragment length polymorphisms, or RFLPs) for dierences by Southern blotting. Those segments containing mutation(s) at or near a splice site are identiable because they were not cut by a restriction enzyme, and are therefore longer, appearing higher on the Southern blot gel. (Longer fragments do not migrate as quickly or as far as shorter fragments.) This technique is used in genetic testing for sickle cell anaemia (Figure 10). Sequencing of restriction enzyme products DNA sequences that have been cut with restriction enzymes can also be sequenced by a specialized amplication technique (Figure 11). Copies of a particular piece of DNA (cut by restriction enzymes) are placed into four vials and amplied by polymerase chain reaction (PCR). As well as the molecular requirements for PCR, each vial also contains a specic, commercially altered nucleotide base (a didioxy derivative). Altered bases are made specic for adenine (A), thymine (T), guanine (G) and cytosine (C). The specic dideoxynucleotide stops amplication each time it is incorporated into the developing chain, instead of its normal nucleotide precursor. Thus, as the sample piece of DNA amplies in each of the vials, in one vial the sequences stop at each A, in one vial they stop at each T, and so on. Between all four vials, the sample piece is cut after each nucleotide, forming fragments that vary by only one base pair. Fragments from the vials, each ending in their particular base, are then allowed to migrate, in parallel, down a Southern blot gel. The shortest fragments travel furthest, the longer segments remain closer to the top. From top to bottom, the banding pattern produced represents fragments that decrease in size by one nucleotide base. The DNA sequence can therefore be read from the shortest, single-base strand at the bottom of the gel, up to the entire sequence length at the top.
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1.3 kb

CCT GAG GAG

MstII

MstII

(a)

CCT GTG GAG HbS: 1.3 kb

SS

AS

AA

1.1 kb

(b)

Lane 1

Lane 2

Lane 3

Figure 10 Direct mutation analysis with restriction enzymes. (a) Sickle cell anaemia and b-haemoglobin gene. Sickle cell anaemia is caused by a basepair substitution in which adenine is changed to a thymine. MstII is restriction enzyme that cuts the specific DNA sequence shown in the figure. MstII will splice exon I of the HbA; however, MstII will not splice HbS because it does not recognize the restriction site due to the A!T mutation. (b) Southern blot analysis depicts the results of gel electrophoresis of an individual homozygous for the sickle cell mutation (lane 1; sickle cell anaemia), heterozygous for normal and sickle cell (lane 2; sickle cell trait/ carrier status) and homozygous for the normal alleles (lane 3; unaffected, not a carrier).

Dot blot Dot blotting with allele-specic oligonucleotides (ASOs) also lends itself to direct mutation analysis. ASO probes are designed to hybridize with both the controls and the mutations complementary sequences. Control and sample DNA is blotted on to lter paper, and permitted to hybridize with both the ASO containing the mutated sequence and the ASO containing the control sequence. Individuals homozygous for the normal sequence hybridize with the control ASO; those homozygous for the mutation hybridize with the ASO containing the mutated sequence. Heterozygous individuals hybridize with both. Analysis is performed by seeing which blots light up on the lter paper (Figure 12). The length of the given ASO is critical. ASOs must be short enough for easy production, but long enough to yield unique sequences and not hybridize to both the control and test DNA. They are typically 1820 nucleotides long. Regardless of the specic method used, direct mutation analysis is advantageous as recombination (due to sister chromatid cross-over) and uninformative matings do not aect the results. Additionally, practitioners need not delve into the patients extended family history, which may cause

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Prenatal Diagnosis

Splice template DNA with restriction enzyme

Template Primer

3 _____ / CGTTACAGG / 5 _____

Add DNA segment to 4 tubes containing PCR requirements. In each tube, also add a dideoxy (dd_TP) derivative, designed to halt amplification at that point dATP dTTP dGTP dCTP +ddATP Resultant fragments: GCA GCAA Run fragments on acrylamide gel A Sequence complementary to template DNA if read from bottom to top T G C C C T G T A A C G GCAAT GCAATGT G GCAATG GC GCAATGTC GCAATGTCC dATP dTTP dGTP dCTP +ddTTP dATP dTTP dGTP dCTP +ddGTP dATP dTTP dGTP dCTP +ddCTP

familial stress, strain and discomfort, as sample information from a large number of family members is not required. Both these problems may occur in linkage analysis, described below. The prime disadvantage is that the control DNA sequence must be known before study. Moreover, only about 5% of disease-causing mutations aect known restriction sites (thus aecting which disorders may be analysed by restriction enzyme sequencing), and laboratory technique needs to be virtually awless. Even with impeccable technical skill, some preparatory techniques are error-prone (e.g. PCR).

Linkage Analysis
Linkage analysis is a means of indirectly detecting a patients mutation status, when several family members are known to be aected with the same genetic disorder, and when an exact mutation is not known. DNA from aected and unaected family members is analysed for polymorphisms such as microsatellite repeats, restriction fragment length polymorphisms (RFLPs) and variable number tandem repeats (VNTRs). Researchers attempt to identify a common polymorphism between all aected individuals in a family, and a dierent common polymorphism between all unaected individuals. These shared polymorphisms are called linkage phases. (For example, in Figure 13, ab is the linkage phase for unaected individuals, while AB is the linkage phase of aected individuals.) Fetal mutation status may then be learned from linkage analysis, by assessing the fetal linkage phase and seeing if it matches the aected or unaected family members linkage phases.

Complementary strand = GCAATGTCC Template = CGTTACAGG

Figure 11 Sequencing restriction enzymes and polymerase chain reaction (PCR) (Sanger dideoxy method).

Figure 12 Representative dot blot analysis of one of the most common cystic fibrosis mutations (DF508). Individual membranes are hybridized with an end-labelled oligonucleotide probe that detects either the normal sequence (left) or the sequence with the DF508 mutation (right). Individuals amplified DNA samples are each blotted twice, once on each membrane. (Each patients DNA is blotted in the same column and row on each membrane.) Results from the first row of this blot are as follows: 1-1 and 1-5 are both normal, homozygous for the normal sequence. 1-2 is a heterozygous carrier, amplifying on both membranes. Both 1-3 and 1-4 show affected individuals, hybridizing only with the DF508 probe. Courtesy of Kristen Skogerbe, Molecular Laboratory, Swedish Medical Center, Seattle, WA, USA.

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Prenatal Diagnosis

1 a b a A b B 1 a b A B

2 a b A B

3 a a b b 2 A B a b

4 a b

(I)

(II)

1 (III) a b a b A B

2 A B a b

3 a b A B

4 a b A B

5 A B

Male: affected Male: carrier Male: unaffected Map Polymorpism A Disease gene locus Polymorpism B

Female: affected Female: carrier Female: unaffected

and the depletion of a product molecule (B). Clinical manifestations of a particular biochemical disorder may be secondary to the progressive build-up of A, depletion of B, or both, and may not present for weeks, months or years. Thus, although some aected or carrier individuals may be identied at birth by newborn screening programmes, or prenatally by CVS or amniocentesis (if the particular family history indicates that the pregnancy is at risk), others may not be detected until severe neurological and/or physical damage has already occurred. Once clinical symptoms arise, the actual diagnosis requires quantication of the given enzyme and/or the accumulated or depleted substances and/or direct mutation analysis. Treatment is specic to the particular disorder, but always involves acute and long-term therapies, the goals of which are: 1. preventing accumulation, and enhancing excretion, of substance A; 2. supplementing substance B; 3. replacing decient cofactors. Diet modication and control are often required, as well as dialysis and/or pharmacological supplementation of specic vitamin cofactors. To ensure the best clinical outcome, diligent management by both patient (through education and compliance) and a team of healthcare providers is essential.

Figure 13 Linkage analysis. Polymorphisms A and B closely flank the disease gene locus, establishing the linkage phases: AB 5 mutation present; ab 5 mutation absent.

The prime advantage is that a specic gene, genetic sequence or gene product does not need to be known before study. The only molecular requirement is that the genetic disorder has been mapped to a general chromosome locus. Yet there are also clinical and laboratory drawbacks. Multiple aected and unaected family members must be willing to undergo testing to determine their DNA commonality. Even when enough members agree to participate, inherent technical obstacles (such as DNA recombination between the marker and the diseasecausing mutation) may complicate linkage phase results. (Identifying polymorphisms on both sides of the mutation, instead of just one, may help to distinguish if/when recombination occurs (see MAP in Figure 13).) Moreover, it may be dicult to identify polymorphisms that are signicantly dierent between aected and unaected family members.

New Diagnostic Techniques

Fetal cells in maternal circulation
A technique currently being developed for clinical use involves isolating fetal cells from maternal blood to analyse fetal chromosomes and/or DNA. Ordinarily, only a very small number of fetal cells enter the maternal circulation; but once they can be readily identied, they will be accessible for analysis by a variety of techniques, without the risks of complications or miscarriage associated with invasive procedures (CVS and amniocentesis). Studies are currently underway to improve isolation and enrichment techniques, as it remains dicult to obtain adequate fetal blood cells to reliably determine fetal karyotype or test for other abnormalities. Nucleated fetal red blood cells (erythroblasts) are currently the ideal candidates for analysis, although leucocytes and trophoblast cells may also be identied. Such cells are identied in a number of ways, including staining specic for fetal cells, amplifying DNA sequences known to be located only on the Y-chromosome (the male sex chromosome, if a male fetus is present), or identifying traits specic to fetal cells (e.g. thymidine kinase activity is only found in fetal cells). The cells are then enriched by a variety of ow and density

Biochemical Analysis
Metabolic disorders (inborn errors of metabolism), are caused by the absence or abnormal function of an enzyme, leading to the accumulation of a precursor substance (A)
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Prenatal Diagnosis

Figure 14 Spectral karyotype demonstrating duplication of chromosome 9 affixed (arrow) to the bottom of chromosome 4. Further analysis with reverse chromosome banding (see images to the left of each chromosome) allowed for further analysis of the breakpoints. This individual is missing a small segment at the bottom of chromosome 4 (the q arm of the chromosome), and has a duplication for the top of chromosome 9 (the p arm of the chromosome). Results: 46,XY,der(4)t(4;9)(q35.1;p12). A subsequent review of the literature found that this patients developmental delay and clinical features indeed matched those described for duplication 9p syndrome (Jones, 1997). Courtesy of Dr Kent Opheim, Childrens Hospital and Medical Center of Seattle.

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Prenatal Diagnosis

gradient techniques, and separated from maternal cells, and can then undergo chromosome and/or DNA analysis.

Spectral karyotyping
Whereas FISH analysis is typically limited by the number of spectrally distinguishable uorochromes and uorochrome combinations available, spectral karyotyping (SKY) can be used to analyse the entire karyotype at once (Figure 14). Each of the 24 distinct chromosomes (22 autosomes, X and Y) is uorescently labelled, with a unique colour assigned to each. This technique is particularly useful for elucidating the identities of complex chromosome rearrangements from multiple translocations, extra structurally abnormal (marker) chromosomes, and de novo unbalanced structural rearrangements. SKY is typically performed after conventional banding techniques have detected an abnormal or unidentiable chromosome. By highlighting every chromosome with its corresponding uorochrome, the identities of de novo marker or unbalanced chromosomes are revealed. Conventional FISH analysis for the specic centromere and particular chromosome regions is used to follow up, conrming the chromosomes identity and isolating which portions are present or rearranged. SKY is both reliable and expedient; its uses continue to be explored and expanded. This technique may even be applied in future to preimplantation genetic diagnosis, or used in conjunction with fetal cell isolation from maternal circulation. A similar technique, whole chromosome painting, highlights all the material corresponding to a given chromosome. The technique helps elucidate the identities of chromosomes involved in translocations and of unidentiable marker or supernumerary chromosomes. The clinical outcomes of such prenatally diagnosed conditions depend upon the chromosome(s) involved, the specic breakpoints, and whether the result is an unbalanced karyotype, with additional and/or missing genetic material.

and the earlier directive approach is no longer commonplace. At present, genetic counselling describes a process in which individuals risks for genetic disease are elicited, genetic screening and testing options are evaluated, and patients are helped to understand risks and options. The goal is informed patient decision-making. The process involves gathering information by collecting an accurate family and medical history, conrming the diagnosis at hand, performing risk assessment and providing clientcentred, nondirective counselling. Counsellors explore clients risk perceptions and personal values to help them arrive at decisions consistent with their values and beliefs. They provide supportive counselling to families, serve as patient advocates and refer individuals and families to community or state support services.

Ethics
Before screening or testing pregnancies for underlying genetic disorders, it is important to consider the ethics of a given situation. Genetic diagnosis may aect decisions about maintaining or ending a pregnancy, place stress upon the family, and/or provide information that may only be pertinent years into the future. Ideally, couples considering prenatal diagnosis meet with a genetic counsellor before diagnostic testing. In some situations prenatal diagnosis may be of little, no or questionable benet especially if parents would not alter their decisions to maintain or voluntarily terminate a pregnancy and/or if treatment is not available for the given disorder. Medical professionals should be aware that it may not be necessary, nor ethical, to diagnose prenatally adult-onset conditions. Moreover, provision of a specic diagnosis may aect the individuals access to healthcare and ability to maintain health insurance for years to come. It is important to explore these and other issues in depth before physically and socially invasive tests are performed. Hospital ethics boards may provide valuable insight for specic cases.

Genetic Counselling
Sheldon Reed rst coined the term genetic counselling in 1947 (Reed, 1955), although its meaning has changed dramatically since then. This profession was rst based in the eugenics movement: counsellors collected data about families, and then advised clients about reproduction to improve the physical and mental capabilities of future generations. Such advice was often biased by social or political agendas, and ultimately led to historical atrocities, such as calling for mentally defective individuals to be sterilized. Thankfully, genetic counselling has changed,
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References
Abruzzo MA and Hassold TJ (1995) The etiology of nondisjunction in humans. Environmental and Molecular Mutagenesis 25 (supplement 2): 3847. Burton BK, Schulz CJ and Burd LI (1992) Limb anomalies associated with chorionic villus sampling. Obstetrics and Gynecology 79: 726730. Ferguson-Smith and Yates RW (1984) Maternal age specic rates for chromosome aberrations and factors inuencing them: report of a collaborative European study on 52 965 amniocenteses. Prenatal Diagnosis 4 (Spec. No.): 544. Firth HV, Boyd PA, Chamberlain P et al. (1991) Severe limb abnormalities after chorionic villus sampling at 5666 days gestation. Lancet 337: 762763.

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Gardner RJM and Sutherland GR (1996) Chromosome Abnormalities and Genetic Counseling, 2nd edn. Oxford: Oxford University Press. Hook EB (1981) Rates of chromosomal abnormalities of dierent maternal ages. Obstetrics and Gynecology 58: 282. Hook EB (1990) Chromosome abnormalities in older women by maternal age: evaluation of regression-derived rates in chorionic villus biopsy specimens. American Journal of Medical Genetics 35: 184187. Hook EB and Chamber GM (1977) Estimated rates of Down syndrome in live births by one year maternal age intervals for mothers aged 2049 in a New York State study: implications of the risk gures for genetic counselling and costbenet analysis of prenatal diagnosis programs. Birth Defects Original Article Series 13(3A): 123141. Jones KL (1997) Smiths Recognizable Patterns of Human Malformation, 5th edn. London: WB Saunders. Moore KL and Persaud TVN (1998) The Developing Human: Clinically Oriented Embryology, 6th edn. London: WB Saunders. Reed S (1955) Counselling in Medical Genetics. London: WB Saunders. Sherman SL, Petersen MB, Freeman SB et al. (1994) Nondisjunction of chromosome 21 in maternal meiosis I: evidence for a maternal-age dependent mechanism involving reduced recombination. Human Molecular Genetics 3: 15291535. Tongsong T, Wanapirak C, Kunavikatikul C et al. (2000) Cordocentesis at 1624 weeks of gestation: experience of 1320 cases. Prenatal Diagnosis 20: 224228.

Further Reading
Baker DL, Schuette JL and Uhlmann WR (1998) A Guide to Genetic Counselling. Brisbane: Wiley-Liss. [A basic overview of topics relevant to the genetic counselling profession.] GeneClinics.[www.geneclinics.org] [For clinical synopsis of selected genetic conditions, including manifestations, causative genetic mutation (if known), inheritance patterns and means of treatment and testing.] GeneTests. [www.genetests.org] [Lists a selection of facilities oering clinical and research testing for selected genetic disorders. Includes the means of diagnosis and contacts at each facility. Included facilities provide their own information, and thus are not expressly endorsed by the website. Only available to registered researchers and health professionals.]

Genetic Alliance.[www.geneticalliance.org] [Provides support group information for selected genetic disorders and advocates for consumers with genetic disorders.] Harper JC, Delhanty JDA and Handyside AH (2000) Preimplantation Genetic Diagnosis. Chichester: Wiley. [A guide to preimplantation diagnosis.] Harper PS (1998) Practical Genetic Counselling, 5th edn. Oxford: Butterworth-Heinemann. [Provides basic information regarding genetic counseling for specic inheritance patterns, disorders and indications. Includes risk assessment. A useful introductory resource.] Hook EB, Cross PK, Schreinemachers DM et al. (1983) Chromosomal abnormality rates at amniocentesis and liveborn infants. JAMA 249: 2043. Jones KL (1997) Smiths Recognizable Patterns of Human Malformation, 5th edn. London: WB Saunders. [A comprehensive guide to human genetic diseases and associated dysmorphology. Many photographs throughout. Resource intended for clinical use.] March of Dimes. [www.modimes.org] [Provides basic, patient-friendly information regarding selected genetic disorders.] Milunsky A (1998) Genetic Disorders of the Fetus: Diagnosis, Prevention and Treatment, 4th edn. Baltimore: Johns Hopkins University Press. Online Mendelian Inheritance in Man (OMIM). [www.ncbi.nlm.nih.gov] [Public database of Mendelian traits and disorders in humans. Each entry has an overview of clinical manifestation and genetic basis of disease, if known. Regularly updated. Technical language used. Intended for clinical use.] Rimoin DL, OConnor JL and Ryeritz RE (1997) Emery and Rimoins Principles and Practice of Medical Genetics, 3rd edn. New York: Churchill Livingstone. [A clinical synopsis of many genetic disorders, including clinical features, genetics and management. Technical language used. Intended for clinicians use.] Saraiya M, Berg CJ, Shulman H, Green CA and Atrash HK (1999) Estimates of the annual number of clinically recognized pregnancies in the United States (19811991). American Journal of Epidemiology 149: 10251029. Thompson MW, McInnes RR and Thompson HF (1991) Genetics in Medicine, 5th edn. London: WB Saunders. [A useful introductory resource to inheritance patterns, risk assessment, prenatal diagnosis, and various classications of genetic disease.] Watson JD, Gilman M, Witkowski J, Zoller M and Witkowski G (1992) Recombinant DNA. New York: Freeman. [A comprehensive textbook that covers the basics of molecular biology. Slightly outdated: many of the basic techniques are still used, but have been modied.]

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