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Prenatal Diagnosis Secondary article Victoria Binns, Swedish Medical Center, Seattle, Washington, USA Nancy Hsu, Swedish Medical

Prenatal Diagnosis

Secondary article
Secondary article

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.

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 significant 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 manage- ment. 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 profes- sionals and other specialists to ensure maximum options and most appropriate care.

Indications for Prenatal Diagnosis

Article Contents . Introduction Indications for Prenatal Diagnosis Methods of Prenatal Diagnosis Chromosome Analysis Karyotype Analysis
Article Contents
. Introduction
Indications for Prenatal Diagnosis
Methods of Prenatal Diagnosis
Chromosome Analysis
Karyotype Analysis
Fluorescence in situ Hybridization (FISH)
DNA Molecular Analysis
Linkage Analysis
Biochemical Analysis
New Diagnostic Techniques
Genetic Counselling
Ethics
.
.
.
.
.
.
.
.
.
.
.

carried during the first 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 partner’s gamete is chromosomally normal). Evidence suggests that lack of recombination between homologues predisposes chromo- some 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%

Advanced maternal age

One of the most common indications for prenatal diagnosis is advancing maternal age (Table 1); as a woman’s age increases, so too does the risk for chromosome aneuploidy in the fetus (Figure 1). While aneuploid conceptions are not uncommon, the majority are mis-

Table 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 findings

.

Abnormal maternal serum screen results

15 14 13 12 11 10 9 8 7 6 5 4 3 2 1 0
15
14
13
12
11
10
9
8
7
6
5
4
3
2
1
0
24
28
32
36
40
44
48
Maternal age
Figure 1
Risk of chromosomal abnormalities by maternal age. Adapted
from Ferguson-Smith and Yates (1984), Hook and Chamber (1977), Hook
(1981 and 1990).
% Risk
Prenatal Diagnosis
Prenatal Diagnosis
Prenatal Diagnosis 46 46 23 23 23 23 23 23 23 23 24 22 23 23

46

Prenatal Diagnosis 46 46 23 23 23 23 23 23 23 23 24 22 23 23

46

23 23 23
23
23
23

23

23

23

23 23 24
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 chromo- somes. 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 significantly increased perinatal mortality due to multiple congenital anomalies, whereas sex chromosome aneuploidies (XXY, XYY, XXX) have highly variable prognoses, affecting fertility, physical appearance and mental status (Gardner and Sutherland, 1996). Although rare, complete nondisjunction of all chromo- some pairs in either the egg (diandry) or sperm (digyny) may also occur, and result in a triploid (3n 5 69 chromo- somes) conceptus, which is associated with high prenatal mortality. Amniocentesis and chorionic villus sampling (CVS) are techniques for obtaining fetal karyotype. Although in- creased 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 transloca- tions, inversions, insertions, deletions or rings.

Information regarding the natural history and prognosis of particular aneuploidies and chromosomal rearrange- ments 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 significantly increased following the birth of an affected child. For women under 35, the recurrence risk is 1% (Gardner and Sutherland, 1996). For women over 35, the recurrence risk resets to the mother’s 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 pregnan- cies. Couples with a history of unexplained loss may,

Prenatal Diagnosis
Prenatal Diagnosis
Prenatal Diagnosis Figure 3 (a) Normal karyotype. (b) Karyotype of an individual with Down syndrome, characterized

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.

Prenatal Diagnosis
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 unaffected. If the rearrangement does cause genetic information to be lost and/or gained, the karyotype is described as ‘un- balanced’: the individual is phenotypically affected (dis- plays signs and/or symptoms). Parents with balanced chromosome arrangements are at risk of having children with unbalanced rearrangements.

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 affected: analysis of family history is often used to assess the suspected disorder’s inheritance pattern and thus accurately assess risk to the fetus. Confirmed 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 specific genetic conditions due to lack of migration, genetic drift or heterozygote advantage within the given popula- tion 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 affected with recessive conditions. Population screening has been developed to screen members of at-risk populations for particular gene mutations: yet sensitivity and specificity 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 identifiable. (patients should be informed of the sensitivity and specificity 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 non- Ashkenazi background; therefore enzyme analysis is more accurate and appropriate.

Teratogens

Maternal disease (e.g. insulin-dependent diabetes, mater- nal phenylketonuria), infection (e.g. toxoplasmosis, rubel- la) or exposure to internal or external substances (e.g. medications, alcohol, radiation) are not associated with

chromosomal or genetic disease, but may lead to fetal abnormality, distress or demise. Prenatally, abnormal ultrasound findings may suggest maternal exposure; if this is suspected or identified during pregnancy, specific management may be offered throughout. Information required to investigate the specific terato- genic effects 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 affect the fetus, or be transmitted in high concentration in breast milk. . Timing and duration of exposure. Teratogens often have the greatest adverse effect during a specific period of embryonic development. . Dosage. Teratogens often only cause detrimental effects when present above a threshold amount. (Unfortu- nately, a given substance’s 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 substance’s effect upon a fetus is difficult, 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 effective dosage is ideal; however, it is important to weigh maternal well-being against the potential for fetal terato- genesis. (For example, if a pregnant woman is at risk of grand mal seizures when not medicated, the benefit 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.)

Abnormal ultrasound findings

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

Abnormal maternal serum screen results

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

Prenatal Diagnosis
Prenatal Diagnosis

Methods of Prenatal Diagnosis

Chorionic villus sampling

CVS was first 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 fluorescence in situ hybridization (FISH)) or assay for biochemical disease. It is typically performed at 10–12 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 undifferentiated cell), before it further develops and forms several cell lineages (Figure 4). In its initial stages, this cell begins dividing, first becoming a morula (ball) of 12–15 undifferentiated 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 differentiate 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 reflect the fetal karyotype. Ideally, 10–20 mg of villi are withdrawn with each sample, using a needle or catheter filled with tissue culture medium. The turnaround time for CVS results depends upon which cell line is analysed. Trophoblast cells (the first to differentiate from the morula) may be analysed directly (same day) or after short-term culture (1–2 days). The villus core, containing the capillaries, is used for long-term culture, as it comprises further-differentiated cells (repre- senting the extraembryonic mesoderm). Long-term culture results, available after approximately 2 weeks, are often used to confirm those of short-term culture. Early studies correlated CVS with fetal limb reduction defects (Firth et al., 1991). Numerous subsequent investi- gations have failed to completely confirm 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 time- frame, the risk may be increased (see review by Burton et al., 1992).

Amniocentesis

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

d’ c’ Trophoblast CVS (chorionic direct ectoderm) preparation b c 1 Chorionic b c d’ CVS
d’
c’
Trophoblast
CVS
(chorionic
direct
ectoderm)
preparation
b
c
1
Chorionic
b
c
d’
CVS
mesoderm
c’
culture
? Fetal blood
2
ys
Amniotic
hy
ys
mesoderm
3
bcd
Amniotic
ep
ectoderm
Amnio-
4
b
centesis
Ectoderm
Mesoderm
? Fetal blood
Ectoderm
Fetal
ps
biopsy
5
b

Undifferentiated cell

Extraembryonic ectoderm

  • Extraembryonic mesoderm

  • Embryonic ectoderm or endoderm

  • Embryonic mesoderm

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 epiblastcells go on to formthe embryo in theinner cell mass. (5)CVS, chorionic villussampling. 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.

Prenatal Diagnosis
Prenatal Diagnosis
Ultrasound probe Chorionic villi Uterine wall Placenta Amniotic fluid
Ultrasound
probe
Chorionic villi
Uterine wall
Placenta
Amniotic fluid

Needle

Chorionic

villi

(a) Ultrasound probe Amniotic fluid Chorionic villi Uterine wall Placenta Biopsy catheter Cervix (b)
(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 fluid contains exfoliated fetal cells that may be cultured to reveal fetal karyotype, and/or perform biochem- ical testing and molecular analysis. Additionally, because amniotic fluid 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 fluid suggest the

presence of such birth defects; if identified, additional screening by fetal ultrasound and for the presence of acetylcholinesterase (AChE) in amniotic fluid may clarify whether an open neural tube or ventral wall defect is present.

Procedure

Under ultrasound guidance, a needle is inserted through the mother’s abdomen and uterus into the amniotic sac,

Prenatal Diagnosis
Prenatal Diagnosis
Ultrasound probe Amniotic fluid Fluid Placenta α-Fetoprotein Acetylcholinesterase Centrifuge Cells Biochemical studies DNA studies
Ultrasound
probe
Amniotic fluid
Fluid
Placenta
α-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 difficult, 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 1–2 % of CVS samples reflect confined 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 offered . 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 fluid are withdrawn (Figure 6). Fetal cells are isolated from the fluid and cultured for analysis. While karyotype results are typically available in 10–14 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 differences 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

Prenatal Diagnosis
Prenatal Diagnosis
Fetal blood Fetal blood Ultrasound probe Umbilical vein Placenta Umbilical Umbilical cord arteries Uterine wall
Fetal blood
Fetal blood
Ultrasound
probe
Umbilical vein
Placenta
Umbilical
Umbilical cord
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 mother’s abdomen to obtain a fetal blood sample. The procedure-related fetal loss rate is estimated at 1–2% (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.

Preimplantation genetic diagnosis and preconception testing

Preimplantation genetic diagnosis (PGD) has been prac- tised 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 unaffected – therefore, the mutation has segregated to the polar body, not to the developing ovum. Once an egg is found to be unaffected, it is fertilized via traditional in vitro fertilization (IVF) and implanted into the uterus. Blastomere PGD first 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

unaffected 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 difficult 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 difficult to amplify) and meiotic crossing-over, which can produce less definitive test results.

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 finding that occurs more commonly in affected than unaffected 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 chromo- some abnormality, cystic fibrosis (CF), intraamniotic haemorrhage, fetal infection, or normal variation. Fol- low-up testing (e.g. amniocentesis) may be offered to rule out or diagnose when abnormal ultrasound suggests an underlying genetic disorder, and/or when family history

Prenatal Diagnosis
Prenatal Diagnosis
Prenatal Diagnosis Figure 8 Ultrasound image demonstrating the measurement of nuchal translucency (the ‘nuchal fold’) at

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

Diagnostic testing

Risk of complications or miscarriage

None

Exists; dependent upon technique:

Amniocentesis: about

 

1/200

Chorionic villus sampling about 1/100

Information provided

Limited

Diagnostic (499% accuracy if gene mutation known)

Timing of results

Instantaneous

Results available in days to weeks

suggests an increased risk. Approximately 50% of fetuses with Down syndrome can be identified by ultrasound by detection of markers such as increased nuchal translu- cency, shortened femoral and/or humeral length, identifi- cation of an echogenic cardiac focus, hyperechogenic bowel, and/or dilation of cerebral ventricles (Figure 8). Ultrasound screening is not diagnostic; moreover, abnormal findings may be transient, affected fetuses may not have detectable anomalies, and unaffected fetuses may show sonographic markers, simply as a matter of normal variation. Ultrasound may identify the presence of a specific diagnosis, such as spina bifida, anencephaly or dwarfism, but may not be able to diagnose the severity of the spina bifida or the specific form of dwarfism. When such an anomaly is diagnosed on ultrasound, pedigree and DNA analysis may be able to pinpoint the specific diagnosis.

It is important for parents to understand the differences between screening and diagnostic testing (Table 3). Screen- ing measures such as ultrasound pose no risk to the pregnancy but findings 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

Prenatal Diagnosis
Prenatal Diagnosis

gonadotrophin (HCG) and unconjugated oestriol (UE 3 ) are measured between 15 and 18 weeks gestation. These substances are of fetal origin and cross from the amniotic fluid into maternal circulation via the placenta. Substance levels, maternal age, weight, race, diabetic status, preg- nancy history and gestational age are taken into account to refine maternal age-related risks. Low maternal serum AFP, low UE 3 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 approxi- mately 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 findings and maternal serum screening is unclear, both techniques can provide couples with more information regarding their pregnancies, and offer 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 first 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.

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 10–15 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 aneuploi- dies. Analysing for the absence, presence, rearrangement, etc. of these bands allows for detection of larger deletions, duplications and structural aberrations. Although G

banding is typically used first to analyse prenatal speci- mens, various other banding techniques (including quina- crine (Q), reverse (R), centromeric heterochromatin (C) and high-resolution banding) may be used to analyse different portions of particular chromosomes (heterochro- matin, nucleolar organizer regions, etc.).

Fluorescence in situ Hybridization (FISH)

FISH is mainly used to detect the presence or absence of microdeletions, microduplications and aneuploidy with- out the full effort associated with DNA sequencing or complete karyotype analysis. This three-step technique allows specific DNA sequences or chromosomes to be visualized microscopically. A specific, single-stranded DNA probe is hybridized to its complementary, target DNA sequence, while the cell is in prophase, metaphase or interphase; fluorescent antibodies are then hybridized to the probe DNA sequence; finally, the fluorescent 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 specific multicoloured centro- meric probes. In fetal trisomies, three probes are present for a specific chromosome, while monosomies show only one. Additionally, different coloured probes may be used in combination in order to analyse multiple chromosomes, while unique sequence probes, specific for a predicted submicroscopic deletion or duplication, can be used to detect the presence of specific monogenic disorders

(Figure 9, Tables 4 and 5).

Prenatal Diagnosis gonadotrophin (HCG) and unconjugated oestriol (UE ) are measured between 15 and 18 weeks

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.

Prenatal Diagnosis
Prenatal Diagnosis

Table 4 Microdeletions/microduplications detectable by fluorescence in situ hybridization (FISH)

Disorder

Chromosome band

Finding a

Angelman syndrome

15q12 (maternal)

Del

Beckwith–Wiedemann syndrome

11p15

Dup

Charcot–Marie–Tooth disease

17p11.2

Dup

Choroideraemia

Xq21.1

Del

Cri du chat syndrome

5p 16

Del

DiGeorge syndrome

22q12

Del

Duchenne muscular dystrophy

Xp21.2

Del

Grieg cephalopolysyndactyly

7p13

Del

Kalmann syndrome/ichthyosis

Xp22.3

Del

Miller–Dieker syndrome

17p13

Del

Prader–Willi syndrome

15q12 (paternal)

Del

Retinoblastoma

13q14.11

Del

Rubinstein–Taybi syndrome

16p13.3

Del

Smith–Magenis syndrome

17p11.2

Del

Shprintzen syndrome/VCFS

22q11.2

Del

α-Thalassaemia

16p13.3

Del

WAGR syndrome

11q13

Del

Williams syndrome

7q11.23

Del

Wolf–Hirschhorn syndrome

4p16

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

Identifies

Centromeric probe

Unique sequence probe Whole chromosome paints Reverse paints Comparative genomic hybridization

The most common aneuploidies (trisomies 13, 18, 21, X and Y) Microdeletions, microduplications Translocations Markers, supernumerary chromosomes Regions of DNA loss or amplification

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 termi- nating a pregnancy. However, FISH analysis for mono- genic disorders is limited because the sequence of the specific 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 sample’s 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 muta- tional analysis appropriate?), how much is known about

Prenatal Diagnosis
Prenatal Diagnosis

the disorder’s molecular genetics and whether other family members are affected.

Direct DNA analysis

Direct mutation analysis involves analysing a target segment of DNA for the presence of a specific mutation. Like FISH, it requires knowledge of the correct sequence for the specific 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 specific splice site) and then analysing resultant DNA fragments (called restriction fragment length polymorphisms, or RFLPs) for differences by Southern blotting. Those segments containing mutation(s) at or near a splice site are identifiable 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 amplifica- tion technique (Figure 11). Copies of a particular piece of DNA (cut by restriction enzymes) are placed into four vials and amplified by polymerase chain reaction (PCR). As well as the molecular requirements for PCR, each vial also contains a specific, commercially altered nucleotide base (a didioxy derivative). Altered bases are made specific for adenine (A), thymine (T), guanine (G) and cytosine (C). The specific dideoxynucleotide stops amplification each time it is incorporated into the developing chain, instead of its normal nucleotide precursor. Thus, as the sample piece of DNA amplifies 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.

HbA: normal haemoglobin gene MstII

MstII

 

MstII

 

Exon I

 

CCT GAG GAG

   
 

HbA: 1.1kb

 

HbS: sickle cell haemoglobin MstII

Exon I

MstII
MstII

MstII

 

CCT GTG GAG

   
 

HbS: 1.3kb

 

(a)

 

SS

AS

AA

1.3kb

1.3kb
1.3kb
 

1.1kb

1.1kb
 
1.1kb
 

(b)

Lane 1

Lane 2

 

Lane 3

Figure 10 Directmutationanalysiswith restriction enzymes.(a) Sickle cell anaemia and b-haemoglobin gene. Sickle cell anaemia is caused by a base- pair 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-specific oligonucleotides (ASOs) also lends itself to direct mutation analysis. ASO probes are designed to hybridize with both the control’s and the mutation’s complementary sequences. Control and sample DNA is blotted on to filter 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 hybri- dize 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 filter 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 18–20 nucleotides long. Regardless of the specific method used, direct mutation analysis is advantageous as recombination (due to sister chromatid cross-over) and uninformative matings do not affect the results. Additionally, practitioners need not delve into the patient’s extended family history, which may cause

Prenatal Diagnosis
Prenatal Diagnosis
Splice template Template 3’ / CGTTACAGG / DNA with restriction enzyme Primer 5’ _____ Add DNA
Splice template
Template
3’
/ CGTTACAGG /
DNA with
restriction enzyme
Primer
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

dATP

dATP

dATP

dTTP

dTTP

dTTP

dTTP

dGTP

dGTP

dGTP

dGTP

dCTP

dCTP

dCTP

dCTP

+ddATP

+ddTTP

 

+ddGTP

 

+ddCTP

Resultant fragments:

 
 

GCA

GCAAT

 

G

GC

GCAA

GCAATGT

GCAATG

GCAATGTC

 

GCAATGTCC

Run fragments on acrylamide gel

Run fragments on acrylamide gel

Run fragments on acrylamide gel
Run fragments on acrylamide gel
 

A

T

G

C

Sequence

Sequence

C

 

complementary to

C

T

template DNA if read

G

from bottom to top

T

   

A

A

C

G

Complementary strand

= GCAATGTCC

 
 

Template

= CGTTACAGG

 

Figure 11

Sequencing restriction enzymes and polymerase chain

reaction (PCR) (Sanger dideoxy method).

familial stress, strain and discomfort, as sample informa- tion 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 affect known restriction sites (thus affecting which disorders may be

analysed by restriction enzyme sequencing), and labora- tory technique needs to be virtually flawless. 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 patient’s mutation status, when several family members are

known to be affected with the same genetic disorder, and

when an exact mutation is not known. DNA from affected

and unaffected family members is analysed for polymorph-

isms such as microsatellite repeats, restriction fragment

length polymorphisms (RFLPs) and variable number

tandem repeats (VNTRs). Researchers attempt to identify

a common polymorphism between all affected individuals

in a family, and a different common polymorphism

between all unaffected individuals. These shared poly-

morphisms are called ‘linkage phases’. (For example, in Figure 13, ‘ab’ is the linkage phase for unaffected individuals, while ‘AB’ is the linkage phase of affected individuals.) Fetal mutation status may then be learned from linkage analysis, by assessing the fetal linkage phase and seeing if it matches the affected or unaffected family members’ linkage phases.

Prenatal Diagnosis Splice template Template 3’ / CGTTACAGG / DNA with restriction enzyme Primer 5’ _____

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 patient’s 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.

Prenatal Diagnosis
Prenatal Diagnosis
1 2 3 4 a a A a A a a a (I) b b B
1
2
3
4
a
a
A
a
A
a
a
a
(I)
b
b
B
b
B
b
b
b
1
2
a
A
A
a
(II)
b
B
B
b
1
2
3 4
5
(III)
a
a
A
A
a
a
A
a
A
A
b
b
B
B
b
b
B
b
B
B
Male: affected
Female: affected
Male: carrier
Female: carrier
Male: unaffected
Female: unaffected
Map
Polymorpism A
Disease gene locus
Polymorpism B
Figure 13
Linkage analysis. Polymorphisms A and B closely flank the

disease gene locus, establishing the linkagephases: AB 5 mutation present; ab 5 mutation absent.

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 affected or carrier individuals may be

identified 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 quantifica-

tion of the given enzyme and/or the accumulated or depleted substances and/or direct mutation analysis. Treatment is specific 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 deficient cofactors.

Diet modification and control are often required, as well as

dialysis and/or pharmacological supplementation of spe-

cific 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.

The prime advantage is that a specific 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 affected and unaffected 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 disease- causing 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 difficult to identify polymorphisms that are significantly different between affected and unaffected family members.

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)

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 identified, 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 difficult 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 tropho- blast cells may also be identified. Such cells are identified in a number of ways, including staining specific 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 specific to fetal cells (e.g. thymidine kinase activity is only found in fetal cells). The cells are then enriched by a variety of flow and density

Prenatal Diagnosis
Prenatal Diagnosis
Prenatal Diagnosis Figure 14 Spectral karyotype demonstrating duplication of chromosome 9 affixed (arrow) to the bottom

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 patient’s developmental delay and clinical

features indeed matched those described for duplication 9p syndrome (Jones, 1997). Courtesy of Dr Kent Opheim, Children’s Hospital and Medical Center of Seattle.

Prenatal Diagnosis
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 fluorochromes and fluoro- chrome 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 fluorescently labelled, with a unique colour assigned to each. This technique is particularly useful for elucidating the identities of complex chromosome rearrangements from multiple transloca- tions, extra structurally abnormal (marker) chromosomes, and de novo unbalanced structural rearrangements. SKY is typically performed after conventional banding techniques have detected an abnormal or unidentifiable chromosome. By highlighting every chromosome with its corresponding fluorochrome, the identities of de novo marker or unbalanced chromosomes are revealed. Con- ventional FISH analysis for the specific centromere and particular chromosome regions is used to follow up, confirming the chromosome’s 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 unidentifiable ‘marker’ or supernumerary chromosomes. The clinical outcomes of such prenatally diagnosed conditions depend upon the chromosome(s) involved, the specific breakpoints, and whether the result is an unba- lanced karyotype, with additional and/or missing genetic material.

and the earlier directive approach is no longer common- place. 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, confirming the diagnosis at hand, performing risk assessment and providing client- centred, 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 affect 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 benefit 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 specific diagnosis may affect the individual’s 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 specific cases.

Genetic Counselling

Sheldon Reed first coined the term ‘genetic counselling’ in 1947 (Reed, 1955), although its meaning has changed dramatically since then. This profession was first 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,

References

Abruzzo MA and Hassold TJ (1995) The etiology of nondisjunction in humans. Environmental andMolecularMutagenesis 25 (supplement 2):

38–47.

Burton BK, Schulz CJ and Burd LI (1992) Limb anomalies associated with chorionic villus sampling. Obstetrics and Gynecology 79:

726–730.

Ferguson-Smith and Yates RW (1984) Maternal age specific rates for chromosome aberrations and factors influencing them: report of a collaborative European study on 52 965 amniocenteses. Prenatal Diagnosis 4 (Spec. No.): 5–44. Firth HV, Boyd PA, Chamberlain P et al. (1991) Severe limb abnormalities after chorionic villus sampling at 56–66 days’ gestation. Lancet 337: 762–763.

Prenatal Diagnosis
Prenatal Diagnosis

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 different 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:

184–187.

Hook EB and Chamber GM (1977) Estimated rates of Down syndrome in live births by one year maternal age intervals for mothers aged 20–49 in a New York State study: implications of the risk figures for genetic counselling and cost–benefit analysis of prenatal diagnosis programs. Birth Defects Original Article Series 13(3A): 123–141. Jones KL (1997) Smith’s 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: 1529–1535. Tongsong T, Wanapirak C, Kunavikatikul C et al. (2000) Cordocentesis at 16–24 weeks of gestation: experience of 1320 cases. Prenatal Diagnosis 20: 224–228.

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 muta- tion (if known), inheritance patterns and means of treatment and testing.] GeneTests. [www.genetests.org] [Lists a selection of facilities offering 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 con- sumers 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 specific 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) Smith’s 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, O’Connor JL and Ryeritz RE (1997) Emery and Rimoin’s 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 (1981–1991). American Journal of Epidemiology 149:

1025–1029.

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 classifications 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 modified.]