When is Prenatal Diagnosis Done ? | Genetics

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Prenatal diagnosis by amniocentesis is useful for prevention of birth of genetically abnormal children. Families having repeated abortions and/or congenital malformations (defects from birth) are likely to be carriers of a genetic anomaly (“high risk” families).

The genetic counsellor can estimate the probability or chance for a future child in such a family to have a genetic defect. But probabilities are not certainties, and after conception the anxiety of the family grows regarding the normalcy of the foetus.

As fetal cells in amniotic fluid of pregnant mothers originate from foetal membranes, skin, respiratory and digestive tracts, their study provides definite information on whether the foetus is actually affected or not. If positive results are obtained by amniocentesis, then termination of pregnancy can be suggested. As there is no cure for most genetic defects after the child is born, the importance of this technique is obvious.

Sometimes families are encountered that have children of only one sex, either all boys or girls. Such parents are desirous of having a child of the other sex. Amniocentesis can determine the sex of the unborn child so that the parents can decide whether or not to continue the pregnancy. In practice however, amniocentesis should not be encouraged for choosing the sex of the child.

Situations:

Prenatal diagnosis by amniocentesis is advisable when parents are seeking genetic counselling in any of the following situations:

1. Previous birth of a child with trisomy or increased maternal age at the time of conception. When there is a trisomic child in the family, there is 1-2 % recurrence risk of another child being born with trisomy. The incidence of trisomy is also related to increased maternal age (above 37 years). It has been demonstrated that increased paternal age (above 55 years) also increases risk for trisomy.

2. If one of the parents is carrying a balanced chromosomal translocation, there is greater risk for producing a chromosomally abnormal fetus. The carrier parent produces unbalanced gametes that could lead to infertility, abortions and abnormal offspring.

About 8-10% of couples who have had multiple recurrent abortions are found to be carriers of balanced rearrangements. It is more serious if the chromosomal rearrangement is caused by a small fragment, because smaller degrees of imbalance are more likely to result in viable offspring.

3. When the woman is a carrier of a deleterious X-linked gene, there is a 50% chance that a male foetus would be affected. In such cases amniocentesis is performed to determine foetal sex. If it is a male foetus, abortion can be considered.

A few of the X-linked disorders affecting males can be diagnosed through enzyme assays, for example Lesch-Nyhan syndrome, Fabry disease and Hunter’s syndrome. In others such as haemophilia and muscular dystrophy (Duchenne type), diagnosis cannot be done antenatally, and, even if the foetus is male, it has equal chances for being normal or affected.

4. When parents are heterozygous for a recessive metabolic defect which can be detected from cultured amniotic cells. More than 40 disorders have been identified so far. The culture technique usually takes 4-8 weeks to grow sufficient cells for enzyme analysis. This is a long time for the family to wait for the result.

Therefore sensitive micro-methods for enzyme assay using micro-spectrophotometry and micro-fluorometry have been applied to prenatal diagnosis. These methods can detect Fabry disease, gangliosidosis and Pompe disease within 2 weeks after amniocentesis.

5. When there has been a previous birth of a child with a neural tube defect such as spina bifida or anencephaly. The levels of alpha-1-fetoprotein in amniotic fluid and maternal serum are higher when the fetus has a neural tube defect as compared to normal pregnancies. If a parent has a neural tube defect there is 2-5% risk of having a child with a similar defect.

6. When parents have had a consanguineous marriage and a recessive disorder is known to have occurred in a relative.

7. When a parent carries a deleterious gene and is also heterozygous for a closely linked marker. The use of marker genes whose products can be detected in the amniotic fluid, when these genes are linked to genetic disorders has been suggested by McKusick and Ruddle (1977).

In such cases the transmission of a genetic disorder to the fetus is determined indirectly through the transmission of the marker gene. This has been done for myotonic dystrophy. The gene for this disorder is linked to the secretor gene which controls the secretion of the blood group substance.

8. When a parent is carrying a mutation for sickle cell anaemia or thalassaemia, it is possible to identify the defect at the level of the genes by the recent DNA sequence and molecular hybridisation techniques. The details are described later.

The procedure for trans-abdominal amniocentesis is as follows. In the 16th to 20th week of pregnancy about 5-10 ml of amniotic fluid is drawn by puncturing the uterus with a hypodermic needle. The foetal cells in amniotic fluid are analysed cytogenetically or biochemically as necessary.

For determination of foetal sex, cell smears are made directly. The slides are stained with Giemsa for Barr bodies, or with quinacrine for fluorescent Y bodies. For confirmation of foetal sex however, karyotype analysis is required. For this, amniotic cells are cultured for 10-15 days and metaphase spreads prepared.

Karyotypes are made from a number of different clones for accurate diagnosis. For correct identification of small deletions and translocations involving small fragments, chromosomes are stained by the banding technique. The sex chromosomes determine foetal sex.

Prenatal Diagnosis by DNA Sequencing Techniques:

In the present era of DNA sequencing techniques, prenatal diagnosis has become possible at the level of the gene. The recent approaches make use of restriction enzymes which cut DNA at specific short nucleotide sequences. Combined with gel electrophoresis for separating DNA fragments on the basis of molecular weight, the technique is useful for characterizing specific DNA fragments from nucleated foetal cells.

The fragments are identified by preparing probes of complementary DNA or RNA and hybridising the probe with the DNA fragment. Since the sequence of the probe is known, that of the DNA fragment is easily determined.

It should be noted however, that probes are not available for all known sense. So far probes for human globin genes only are obtainable. Such probes can be useful for prenatal diagnosis only if the fetus carries a risk for an alteration in the sequence of a globin gene.

How exactly restriction enzymes are utilised for prenatal diagnosis can now be explained. The presence of alterations in the DNA sequence of a mutant gene in the foetus changes the pattern of restriction endonuclease recognition sites. For example the restriction endonuclease Mnl 1 normally recognises the sequence GAGG.

In a fetus carrying a mutation for sickle cell haemoglobin the sequence 5′-GAGG-3′ in the β globin gene changes to 5′-GTGG-3′ which the endonuclease Mnl 1 is not able to recognise. Such a fragment is detected because it is deleted. However, due to the small size of fragments generated by Mnl 1, there are technical difficulties in antenatal diagnosis of sickle cell anemia by this method.

An alternative approach for prenatal detection of sickle cell anaemia has been developed by Kan and Dozy (1978). The method is based on detecting a change in the DNA sequence within a region flanking (lying adjacent to) the β globin gene. This became possible when it was found that the sickle cell mutation was frequently linked to an altered DNA sequence in the flanking gene.

The endonuclease Hpa I recognises the normal sequence in the flanking region which is located 5,000 bases downstream from the β globin gene. When this sequence is altered, as in the case of homozygous sickle anaemia, Hpa I is not able to recognise the site and a fragment is deleted. In man the globin genes contain repeated DNA sequences. There are two α globin genes on chromosome 16, while the genes for β, δ and γ chains are on chromosome 11.

It is known that crossovers occur within human globin genes so that some sequences within or between the globin genes become deleted. Such deletions have been found to produce clinical disorders such as haemoglobin H disease, hydrops foetalis, the thalassaemias and HPFH.

In the case of α-thalassaemia, the α chains of haemoglobin are not synthesised due to deletion of all the four α chain genes from DNA. Using complementary RNA probes and hybridisation technique on cultured amniotic cells, Kan and Dozy were able to detect a-thalassaemia-1 where 2 genes are deleted and haemoglobin H disease having 3 deleted genes.