Techniques used in Detection of Genetic diseases-Part 1

There are several hundred genetic diseases seen among human beings. Most of them are caused by single-gene recessive mutation. Though most of these diseases are manageable, there is no cure for them except for Gene therapy which is fast emerging.

It is important to first identify the genes responsible for a genetic disorder before establishing the disease. Identification of responsible genes is vital in view of one or more of the following considerations:
1. It may provide information about the biochemical basis for the genetic diseases which in turn enables the development of an appropriate management regime or therapy.
2. Establishing the type of mutation present in the defective gene would the make it possible to develop an appropriate screening programme, by which the normal appearing carriers of the disease could be identified. By identifying the potential carriers, precautions can be taken to reduce the possibility of the disease being expressed. The carriers can be counselled about the risk of their children inheriting the disorder. Foetuses that are detected to carry the genetic disorder can be terminated after counselling.
3. Only after the defective gene is identified, can gene therapy be suggested..

Detection of genetic diseases in foetus:
The occurrence of genetic disorders can be minimised by an early detection of the afflicted foetuses and termination of such pregnancies.

Obtaining the foetal cells:
Earlier methods of obtaining foetal cells involved extraction of amniotic fluid by a method named amniocentesis. Amniotic fluid which contains free foetal cells is withdrawn using a hypodermic syringe. The disadvantage of this method was the amniocentesis could be done only after 18 weeks or later in pregnancy which would be too late for termination. Recent developments allow foetal cells to be extracted during 6 to 8 weeks of pregnancy by performing a biopsy of trophoblastic villi which form the external part of the embryo. This specimen provides sufficient amount of foetal DNA. The foetal cells are recovered from the specimen by centrigugation. If need be these cells can be cultured in vitro for obtaining sufficient cells.

Methods of detecting genetic disorders:

1. Karyotype Analyses:
Establishing the karyotype of cells provides knowledge about syndromes caused by gross chromosomal aberrations.

2. Enzyme assay:
In most genetic disorders, the presence of defective genes results in production of defective proteins or enzymes. Sometimes the enzyme is not produced at all. Most of these proteins have already been identified .Thus an enzyme assay of the foetal cells helps in detecting the genetic disorder.

3. Restriction Fragment Length Polymorphism (RFLP):
Analysis of RFLP makes use of the fact that in certain genetic disorders, the gene mutation changes the recognition site for restriction enzyme. It can either create a new site or delete the normal recognition site .The RFLP thus obtained is detected by southern blotting using a small sequence of the specific gene as the probe. For example the recognition site for the restriction enzyme MstII is "CCTNAGG" in the normal beta globin gene of haemoglobin. In case of sickle cell anaemia the mutation of "GAG" to GTG" deletes this recognition site. DNA of the foetal cells is digested with MstII and electrophoresed on gel and probed with a sequence of the beta globin gene. Parallely normal beta globin gene DNA is also subjected to the same process. If the test sample shows bands comparable to normal DNA it means the test individual has a normal beta globin gene. In case of a sickle cell mutant beta globin gene the pattern of the bands will differ from the normal gene in a detectable way. RFLP can be used only in genetic disorders where the mutation leads to a change in restriction sites and thus is not applicable in detection of all disorders.

4.Oligonucleotide probes.
This is a general approach of detection. This method uses radiolabelled oligonucleotide probes. A set of two probes is used. One probe is complementary to the normal gene and the other has complimentary sequence to the mutant gene. These probes can differentiate the normal and the defective genes using the southern blot technique. Sickle cell anaemia has been detected using these probes. A set of two 19 base long probes have been employed to detects sickle cell anaemia. one of the probe carries complimentary nucleotides to that of a normal beta globin(beta-a) gene while the other has complimentary nucleotides to a mutant beta globin gene(beta-s).The southern blots of normal individuals will hybridize only with beta-a while those of sickle cell homozygote hybridize with beta-s only, and those with heterozygote sickle cell hybridize with both. Another application of this method is the detection of alpha-antitrypsin gene implicated in pulmonary emphysema.
Oligonucleotide polymers can be used as detection tool only when the genetic sequence of both the normal and the mutant gene is known.

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