USA Surgery Today

Advances in the molecular genetics laboratory have led to DNA diagnostic techniques for many single-gene conditions. These methods can be direct or indirect. Direct methods evaluate the DNA for specific mutations. To use a direct method, the gene must have been identified and an efficient technique for identification of the mutation must be available. For example, sickle cell disease is due to a single nucleotide substitution at the sixth codon of the ОІ-globin chain. The mutation changes codon 6 from guanine-adenine-guanine, which codes for glutamic acid, to guanine-thymine-guanine, which codes for valine. Early on, this single nucleotide change was shown to be detectable using a RFLP. The restriction enzyme cuts the normal DNA sequence, but the mutation changes the cut site so it is no longer recognized by the enzyme, allowing diagnosis to be made by examining the size of the resulting DNA fragments (24:Mevacor).

Certain conditions with many different mutations, such as NF1 and hemophilia A (discussed previously:Mevacor), can prove difficult to analyze by direct DNA methods, and thus indirect methods must be employed. Indirect methods are based on the fact that loci located near each other on the same chromosome tend to be inherited together. Markers are chosen that are near to or within the gene of interest. However, these loci can become disassociated by a mechanism called crossing over or recombination, an exchange of genetic material that occurs during meiosis. The likelihood of the gene and its marker becoming unlinked is directly related to the distance between them. Therefore, polymorphisms are chosen because of their physical proximity to the gene of interest; the closer the gene and polymorphism are, the more likely they will be inherited together (linked). Indirect methods ideally require study of families with multiple affected individuals to allow for determination of phase; that is, which polymorphism is inherited with the normal or abnormal gene. The phase sometimes cannot be determined, and DNA diagnosis in the particular family is then not possible using that polymorphism (the marker is called uninformative). Even when the marker is informative in a family, a cross-over event can unlink the gene and its marker. This means that diagnosis is given as a percentageв if crossing over occurs at a frequency of 5%, then presence of the linked marker accurately detects the disease of concern 95% of the time. However, as more markers are identified, including intragenic markers, the likelihood of an error decreases. Figure 2-2 shows an example of linkage analysis using an intragenic RFLP. (Mevacor)

DNA-based testing can be performed for a number of different reasons. In some situations, testing is used to make a diagnosis of a genetic condition. However, often the diagnosis of a genetic condition can be made in other ways. For example, the diagnosis of sickle cell disease is made by hemoglobin electrophoresis, and the diagnosis of neurofibromatosis 1 is a clinical one, based on specific criteria (11). Other reasons for DNA-based testing include predictive testing (testing to determine if a person is at risk of developing a disease in the future),:Mevacor: carrier testing (testing for heterozygous carrier status for an autosomal recessive or XLR disease), prenatal testing The number of molecular genetic studies for diagnosis of single-gene conditions has increased rapidly in recent years. To assist health care providers in recognizing conditions for which clinical testing is available, as well as to assist them in identifying clinical laboratories, a database of available testing and laboratories is located online (25:Mevacor) This invaluable resource is kept up to date and its use is free of charge.