Techniques used in Detection of genetic Diseases - Part 2

Identification of Genes causing Genetic disorders

Most of the techniques used in identifying a gene responsible for a genetic disorder can be applied only when there is pre-information about the genes or the gene products. However in many cases where there is no knowledge about the genes or the gene products a different approach has to be used. In such cases the most probable gene resulting in the disorder can be identified by using other techniques.
Let us take the example of BRCA1 which is responsible for human breast cancer. This gene is nearly 100 kb long and had 22 exons. The strategical methods used in the identification of the gene are as follows:

1. Pedigree analysis:
This is the basic step. Here the pattern of inheritance of the disease is analysed within families which exhibit high incidence of the disease. DNA samples of at least three generations from each family are taken for the study and the number of members per generation should be as large as possible. After a detailed analysis of the DNA, a linkage between the disease and other traits, especially DNA markers, is established.

In case of gene BRCA1, an RFLP locus named D17S74 was found to be associated with breast cancer. Genetic analysis of a considerable number of women with breast cancer showed the same version of D17S74. This locus is situated on the long arm of chromosome 17, representing nearly 20 Mb DNA containing around 1000 genes.

2. Short Tandem Repeats (STR) Linkage Mapping:
Once the marker is determined the next step is to analyse the identified locus and do further fine scale mapping. This is done with the help of short tandem repeats (STR) linkage mapping.
STR linkage gene mapping of BRCA1 showed that it was located in a 600 kb region identified by previously mapped marker loci D17S1321 and D17S1325,However since it is not possible to narrow down to such a short segment of DNA ,a region of 10 Mb or more is explored further.

3. The concluding step is to identify the most probable gene responsible for the genetic disorder and confirm that it is responsible for causing the disorder. This has different approaches. All the genes of the specific locus identified in the previous step are candidate genes and are further analysed to pin point the gene in question

• a.) The candidate genes of a variety of tissues can be analysed using northern blot or RT-PCR. BRCA1 is expected to be expressed in breast and ovarian tissues because ovarian cancer is often linked with inherited breast cancer.

• b.) The candidate genes can be subjected to Zoo blot analysis where it is hybridized with DNA from different species. It is a known fact that most, important human genes usually have homologues in other mammals.BRCA1 was found to have homologues with mice, pigs, Rabbits and sheep, but not with chickens.

• c.) The gene sequences of affected and normal individuals are analysed and compared. The gene BRCA1 showed frame shift and nonsense mutations in five families which had cases of breast cancer.
d.)The identity of the mutated gene can be confirmed by inducing the same mutation in mice. The mice in which the mutation is carried out are termed as Knock out mice. The knock out mice is expected to exhibit the same genetic disorder. A positive result from such a trial confirms the gene in question to be responsible for the specific genetic disorder.

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