The Institute’s twenty-ninth annual symposium was held on 17 & 18 September 1992. The twelve papers will be published in full as a single volume in association with Macmillan Press.
In Britain each year, around 14,000 infants are born with some form of genetic disorder. The limitations of current techniques for prenatal diagnosis mean that only by the abortion of many normal fetuses can these births be avoided. Only better diagnosis can reduce this waste.
Sources of fetal material are:
Diagnostic techniques fall into two categories - microscopic (those involving chromosomal changes which can be identified by a trained cytogeneticist using a microscope) and submicroscopic. The former involve the insertion or deletion of at least five million base pairs of DNA. The latter may be no more than a change in a single base pair resulting in a single amino acid change in the protein for which the gene codes; or it may change a codon for an amino acid into a ‘‘stop’’ codon which causes premature termination of protein construction.
Twenty conditions involving single point mutations have been traced to specific locations and can be investigated clinically. Never is the same mutation responsible for all cases of a disease, so it is useful to conduct a family study to identify the exact mutation which the fetus may have inherited. If the mutation has been isolated as a specific DNA sequence, as in the case of cystic fibrosis, it can be identified directly. In other cases it is necessary to use nearby markers.
In the case of cystic fibrosis, one person in twenty-five has a defective gene and there are over 200 different mutations. It is impractical to search for them all but just one mutation is responsible for 70% of British cases - a deletion of three base pairs causing a single amino acid deletion in the resulting protein.
The technique of polymerase chain reaction (PCR) is used to multiply a tiny sample of DNA by 10-100 million times for testing.
Duchenne muscular dystrophy is an X-linked condition. 60-70% of affected boys have part of the gene deleted and of these 50% lack the middle two million base pairs. If another member of the family has a deletion, it can be sought in the DNA of the fetus under investigation.
A new phenomenon has been discovered this year in connection with the ‘‘Fragile X’’ syndrome, an X-linked condition causing mental retardation. Normal people have up to 60 repeats of the base sequence CGA near the beginning of the responsible gene whereas affected individuals have hundreds or thousands of repeats. Similarly myotonic dystrophy is caused by excessive repeats of CTG near the end of the gene (outside the coding sequence) and the severity of the condition is related to the number of repeats.
Ultrasound scanning is becoming increasingly refined and offers excellent views of the developing fetus. As a result, structural abnormalities can be detected at ever earlier stages of development. But this process has a limit, determined by the time a specific abnormality originates.
It is not feasible to make detailed studies of organogenesis in humans. Fortunately the mouse can be very easily studied and is similar to the human in the overt processes of development.
Neural tube defects - anencephaly and spina bifida - are among the most common congenital malformations affecting children in the United Kingdom. Although it is not known exactly how or why neural tube defects arise, it is believed that the neural folds somehow fail to fuse to form a tube rather than that they fuse and then burst open again. Since this primary form of neurulation is normally completed by day 26, in principle neural tube defects could be detected by imaging techniques from the end of the fourth week of pregnancy.
The upper lip is formed by the fusion and downward movement of the two medial nasal swellings as a result of the two maxillary swellings fusing with their outer parts. Failure of either maxillary swelling to fuse will give rise to lateral cleft lip; failure of the nasal processes to form gives rise to medial cleft lip. Fusion to form the upper lip occurs at the end of the seventh week, so cleft lip cannot be diagnosed before then.
The main part of the palate is formed from two shelf-like outgrowths from the maxillary swellings. Fusion in the midline takes place in the tenth week. Cleft palate occurs when the growth or movement of the palatal shelves is impeded in some way so that fusion does not occur. Thus it does not arise until after ten weeks of development.
There are many congenital abnormalities affecting the eye. Anophthalmia, complete absence of the eye, can be caused in several ways. Cyclopia, that is partial or complete fusion to form a single eye within one orbit, usually has a supervening proboscis but the nose is absent. This malformation would appear to derive from failure of the establishment of two separate cerebral hemispheres rostrally. It occurs very early and is potentially detectable very early on.
The limb buds appear at around 25-26 days. In the sixth week the distal ends of the buds flatten to form paddle-like foot and hand plates. Digital rays develop and digits form by death of cells between the rays. If this programmed cell death does not occur, syndactyly results - ie cutaneous fusion of the fingers or toes. Secondary abnormalities of the limbs are common, for example intra-uterine amputation. This is thought to be the result of constriction by aberrant strands of amnion. The digits should separate and the fingers become distinct in the eighth week, but intra-uterine amputation presumably occurs later.
Mr Barron opened with a review of the changing status accorded to the fetus from ancient times to the present day, remarking how recently its true origin and development had been recognised. Not until 1827 was the mammalian embryo described in detail and not until 1956 was the true number of human chromosomes shown to be 46.
Perceptions of the fetus over the last forty years have been greatly influenced by four important advances in medical science:
Early attempts to image the fetus used X-rays but were limited to conditions affecting the bony skeleton and were abandoned because of the risk of harming the fetus. Ultrasound, developed from the SONAR system used during the Second World War to detect submarines, took a leap forward in 1975 with the introduction of real time imaging and has been further refined using the Doppler frequency shift to demonstrate flow within structures such as the heart. The new technique of magnetic resonance imaging allows the depiction of chemical function but is still difficult to apply.
Survival of infants born as early as 28 weeks of gestation has improved from ten to about fifty percent since 1965. As a result, obstetricians are much more prepared to consider premature delivery where maternal well being is compromised by continued pregnancy, thereby adding to the number of these very small babies.
The first baby conceived as a result of in vitro fertilisation was born in 1978, since when the technique has advanced considerably. It has become possible to examine and even change the zygote before implantation. An embryo can be frozen, stored and later implanted to become a fetus after the death of one or both of its parents. The biological and genetic mothers need no longer be the same person. Law and ethics have not yet caught up with these advances.
The possibilities of gene therapy increase daily as a result of the staggering advances in the understanding of genetics at the molecular level.
Until recently, the peri-implantation stages of development were poorly understood. The advent of in vitro fertilisation has facilitated the study of what happens in the first few days of development and a significant range of genetic abnormality can now be detected at this stage. More traditional techniques, such as CVS and amniocentesis, lead to diagnosis at a stage when the only alternative to the birth of an abnormal baby is a termination which may, for various reasons, be unacceptable. There are therefore attractions in the possibility, in the case of patients known to be at high risk of a specific abnormality, of pre-implantation diagnosis by testing a single cell from the developing embryo. Such a technique will have to meet four criteria:
1. There must be a realistic chance of pregnancy following in vitro fertilisation. IVF pregnancy and live birth rates are around 17% for infertile couples; we do not currently know the rates for fertile women.
2. Techniques for cell removal must be safe and effective. Cells can be taken at different stages of development, each with its own advantages and drawbacks. Ethically, the ideal is a biopsy of the polar body which is not part of the fetus - it is formed during egg production and contains alleles complementary to those in the egg. The technique only establishes which genes the fetus has inherited from the mother and so is limited to homozygous conditions.
3. Probes for the relevant genetic diseases must be available. The assay must be:
4. Diagnostic techniques must be reliable. The presence of maternal mRNA and protein in the egg cytoplasm and the incidence of multinucleated cells are among the causes of error currently encountered.
Genetic diagnosis at pre-implantation stage is now feasible - but still a long way from the 5% error rate which will make it clinically useful. Further progress depends on the opportunity to do research on embryonic material.
Ultrasound scanning is the most common of the non-invasive diagnostic techniques. It is used:
Another non-invasive technique is maternal serum sampling for the detection of alphafetoprotein. This gives good results in the detection of neural tube defects and is also useful for detecting trisomy 21.
Amniocentesis is the safest of the invasive tests and has a wide range of application:
The main drawback of amniocentesis is the time taken to culture the cells before a diagnosis is possible. Samples are now being taken earlier (9-14 weeks’ gestation) but possibly at the cost of some reduction in safety. Advances in cytogenetic technique may soon permit faster results from amniocentesis at the usual time.
Chorion villus sampling permits rapid diagnosis of a wide range of indications but safety trials have given conflicting results. Questions which need answering are:
Fetal blood sampling is used to investigate karyotype, rhesus status, fetal well-being and infections. There is a 1-2% procedure-related fetal loss in an uncomplicated pregnancy; the loss rate is related to the severity of the indications and the duration of the procedure but not to the experience of the physician provided that there is good supervision.
It is important to explain to the mother the reasons for any investigations.
Referral to a fetal anomaly clinic usually occurs at one of three stages during pregnancy:
Maternal age and family history are the main reasons for referral at this stage. It is necessary to carefully balance the risks of anomaly at any particular age against the relative risks of investigation, particularly CVS and amniocentesis. Although CVS presents the higher risk of miscarriage, the earlier date at which it can be carried out (less than 12 compared with over 16 weeks’ gestation) and the shorter time taken to obtain results (24 hours compared with 2 weeks) are significant advantages in many cases.
Many of the conditions diagnosed at this stage present the obstetrician with difficulty in deciding whether to offer termination of pregnancy:
Most maternity units offer a routine antenatal scan at around this stage. The timing is late enough to allow the detection of many structural deformities and early enough for termination to be offered. The obstetrician has to decide whether to offer termination in a variety of difficult circumstances:
3. 28-30 Weeks’ Gestation
One of the most common clinical occurrences leading to referral at this stage is fetal intra-uterine growth retardation. The benefit of diagnosis is that it permits the management of delivery to be planned.
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The second half of this report will appear in the next issue of the Newsletter.
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