Clinical Risk >
Volume 14, Number 6 >
Pp. 218-221
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Prenatal diagnosis |
Email: fiona.macdonald{at}bwhct.nhs.uk
 | Introduction |
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This article will primarily concentrate on aspects of molecular genetics testing and identify what measures are in place in order to prevent an incorrect diagnosis. However as testing relies heavily on collaboration with cytogenetics laboratories some details of cytogenetic analysis are included for clarification.
| Molecular genetics and cytogenetics |
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Box 1 Common single gene disorders for which prenatal testing is carried out
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In the last two to three years molecular genetics has also gradually replaced some areas of prenatal cytogenetics. A molecular test is now the initial test performed to identify the commonest chromosome abnormalities, notably Down's syndrome (trisomy of chromosome 21), Edwards' syndrome (trisomy of chromosome 18) and Patau syndrome (trisomy of chromosome 13). Results can be obtained in 2–3 days, in comparison to 10 days for cytogenetics testing, so gives women rapid reassurance that their fetus does not have any of the three most common abnormalities detected during prenatal testing.1 Further cytogenetic analysis is subsequently carried out for the identification of rarer chromosome anomalies. However, in some regions in the UK this additional chromosome analysis is being phased out for the majority of cases.
| Regulations and best practice |
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Clinical scientists working with genetics laboratories are now all state registered with the Health Professions Council, following a designated training period. Technical staff perform a large proportion of the technical work of laboratories; although not currently state registered, the process of registration for this staff group is underway and awaiting Privy Council approval. A voluntary register has been established in the meantime.
| Quality control and assurance |
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Best practice guidelines for molecular genetic analysis have been established over a number of years by the Clinical Molecular Genetics Society (http://www.cmgs.org). These guidelines make recommendations as to best practice for a variety of genetic conditions analysed at the DNA level as well as best practice for specific techniques, for internal quality assurance and for reporting. They are updated at intervals, usually following a best practice meeting attended by representatives primarily from NHS molecular genetics laboratories. The guidelines are not mandatory but recommend practice which should be implemented to ensure quality of service.
| Molecular genetics of single gene disorders |
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Autosomal conditions are those inherited on chromosomes 1–22 whereas X-linked conditions are carried on the X chromosome. Autosomal dominant conditions affect both men and women and a mutation is only required in one of the two copies of the gene for symptoms to manifest. Offspring of an affected parent have a 50% chance of inheriting the condition. Huntington disease and myotonic dystrophy are examples of autosomally inherited conditions.
In autosomal recessive conditions, an affected individual will have mutations in both copies of the gene. The parents of an affected individual will have a mutation in only one copy of the gene so will be carriers of the condition. Such parents have a 25% risk of having another child with the condition. Cystic fibrosis and spinal muscular atrophy are examples of autosomal recessive diseases.
X-linked dominant conditions also affect both sexes although the condition tends to be less severe in women. Offspring of an affected mother have a 50% chance of developing the condition. All daughters, but no sons, of an affected father will be affected. These conditions are relatively rare; examples include hypophosphatemia and Rett syndrome.
Finally in X-linked recessive disease, it is men who are primarily affected and 50% of male offspring will be affected. Affected boys are usually born to an unaffected mother who is a carrier of the condition. An example of such a condition is Duchenne muscular dystrophy.
Prior to carrying out a prenatal test it is always important to confirm the diagnosis in the affected parent to ensure that they do carry the relevant mutation. Similarly in recessive conditions it is important to confirm parental carrier status. On occasions, when performing carrier testing, non-paternity can be identified. This has to be dealt with through careful genetic counselling as it has obvious bearing on the risks to any subsequent fetus and could lead to unnecessary prenatal testing.
| Sources of genetic material |
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Amniotic fluid is the fluid which surrounds the fetus allowing it freedom of movement and cushioning it during development. It is primarily made up of water but also contains proteins, carbohydrates, enzymes and hormones and, importantly, fetal cells. The volume of fluid increases during pregnancy and by about 15–18 weeks of pregnancy the volume is sufficient to allow safe removal of some fluid under ultrasound guidance with very little likelihood of harming the fetus. The procedure does however carry a small miscarriage risk (less than 1%).
Chorionic villi are part of the extra embryonic tissue and have the same genetic origin as the fetus. Again these can be sampled under ultrasound guidance. The main advantage of CVB over amniocentesis is that the procedure can be carried out from 10–13 weeks allowing diagnosis at an earlier stage and, if required, termination of affected pregnancies in the first trimester. It has a higher miscarriage rate than amniocentesis, namely 1–2%.
Fetal blood samples can be obtained after 18 weeks of pregnancy. Blood samples are removed from the umbilical cord under ultrasound guidance. This procedure tends to be performed on late gestation referrals or where results from amniocentesis or CVB have given ambiguous results.
Preparation of tissue for analysis
A major potential cause of error in prenatal testing is the presence of maternal cells in the sample. This can lead to misdiagnosis through testing DNA originating from maternal cells rather than fetal ones. In amniotic fluid, blood staining of the sample can indicate the possibility of maternal cell contamination (MCC) although it is recognized that in some cases the blood is of fetal origin. Visible blood staining is seen in 1–2% of fluid samples and, following centrifugation to pellet cells, is observed at a much higher frequency of approximately 38% of samples. Sensitive molecular testing methods have identified the presence of a maternal contribution in amniotic fluid in 9.1% of direct or cultured preparations, 17.8% of which had no evidence of visible blood staining.2 This study indicates how important it is to rule out MCC when performing prenatal diagnosis.
When CVB samples are taken, maternal tissue is present. This is removed by careful microdissection under the microscope by an experienced cytogeneticist to leave cells solely of fetal origin. It is important during this procedure that only one patient's sample is handled at any one time to ensure that there is no mix-up of samples. As the procedure of microdissection is extremely rigorous it is rare to find significant levels of MCC in the cleaned material but it is still important to rule out the possibility when carrying out any molecular prenatal test. Methods of MCC detection are described below after the basic methodology used in molecular genetic analysis has been described.
DNA is extracted from either amniotic fluid, CVB or fetal blood prior to molecular analysis. There are many proprietary kits on the market which can be used for DNA extraction and the procedure can be carried out either manually or by an automated system. Whichever method is used, the ideal is to minimize the number of times samples are transferred from one container to another during the extraction process. The use of automation can minimize the possibilities of error during transfer but is generally only of value if significant numbers are being handled (e.g. in case of molecular tests for the common chromosomal trisomies). For many prenatal tests looking for single gene disorders, only one or two tests are carried out at any one time so manual methods of extraction are still used.
Polymerase chain reaction and Southern blotting
The majority of prenatal tests will be carried out using one of two techniques, the polymerase chain reaction (PCR) or Southern blotting. PCR forms the basis for the majority of all work carried out by molecular genetics laboratories. It is a method which allows for the production of multiple copies of a target fragment of DNA which can then be analysed by a variety of downstream processes. The target, in the case of most prenatal tests, will be a region of a gene in which a mutation has been identified in the family under investigation. PCR is an enzymatic reaction in which the target region is faithfully copied using a DNA polymerase enzyme to produce an exact copy of the fragment. This copy is then itself the template for further rounds of amplification (usually around 30) leading to an exponential increase in the number of copies of the specific target. In theory the reaction can start with only one or a few copies of DNA and at the end of the process, millions of copies are produced. This process usually takes around 3 hours to complete. PCR is extremely sensitive, which is why this test can pick up any MCC of the sample. The sensitivity of the technique also means that it is important always to include a negative control when tests are being set up. This tube will contain all the necessary components of the PCR reaction but without any added DNA. This ensures against contamination of any of the reagents which could lead to the amplification of DNA from a source other than the patient under investigation. It is also best practice to include a positive control, usually DNA from the parent or parents of the fetus. This ensures that the mutation present in the parental sample(s) can always be detected by the analysis. Also a DNA sample from a normal individual is included for comparison.
Once the PCR reaction has been completed, the products of the reaction are analysed to look for any abnormality being present. This process will vary according to the disorder and the nature of the causative mutation. Most commonly the analysis will either be a comparison of the size of the PCR fragment in the patient compared to an individual without the mutation or may involve sequencing of the PCR product to determine the specific sequence of DNA bases in the patient in comparison to the positive and normal control samples. The entire process of testing is normally completed within 3 days. This is the Department of Health target reporting time where the analytical method used for prenatal diagnosis is PCR-based.
In the majority of PCR reactions carried out routinely, the fragment which is copied is usually under 1 kilobase (1000 bases of DNA) in length, a size which is easily amplified by PCR. For a small number of genetic conditions however the abnormality results from a significant increase in the size of a region of DNA within a gene. This mechanism is associated with both Fragile X mental retardation and myotonic dystrophy, two of the disorders which most commonly require a prenatal test. In these conditions the abnormal region of the gene is expanded to such an extent that PCR cannot be used to copy the DNA. The alternative technique used in these cases is termed Southern blotting. This is a much longer process than PCR and involves a series of steps, each of which can take up to 24 hours and overall can take 5–10 days to perform. Women must therefore be counselled in advance that they will not receive a rapid result.
As mentioned above, it is important to ensure that MCC is excluded when carrying out a prenatal test. Following a meeting of the Clinical Molecular Genetics Society in 2007, this is now recommended as best practice. Although laboratories did carry out these checks prior to 2007, further guidance was provided as to who should carry out the testing and how it should be done.
MCC testing should be carried out on all prenatal DNA samples, whether from CVB, amniotic fluid or fetal blood. The markers used for this type of analysis are termed microsatellite markers. These markers, of which many thousands are recognized, are located throughout the human genome but are not related directly to any disease as they are found out-with genes themselves. They therefore give no information of disease status but can be used to ‘label’ or ‘tag’ individual chromosomes as they are highly variable in size so can be used to differentiate maternal from paternal chromosomes. This type of marker is routinely used in forensic investigations and paternity studies for similar reasons.
DNA from a maternal blood sample should always be analysed at the same time as the fetal sample. An example of MCC, manifesting as additional peaks on the trace produced following PCR and size separation is shown in Figure 1. If MCC is detected in a sample, this cannot be used for the gene specific test as the result will be compromised. A repeat CVB or amniotic fluid sample may be required or if the initial analysis was carried out on DNA extracted directly from the tissue of origin it may be possible to repeat the analysis on DNA extracted from cells cultured in the cytogenetics laboratory.
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| Auditing prenatal testing |
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| Conclusions |
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| Footnotes |
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| References |
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Introduction
