Human Biochemistry, Volume 1 - Murray R. 1993

Structure and Functions of Proteins and Enzymes
Enzymes: General Properties
Application of Restriction Endonucleases in Diagnostics

The Diagnosis of genetic diseases has received a powerful impetus thanks to recent advances in Recombinant DNA technology. It has long been known that all hereditary disorders are caused by alterations in DNA, but Methods for the direct determination of DNA nucleotide sequences have emerged only recently. For example, based on Hybridization screening of DNA fragments (Southern, 1975), a sufficiently sensitive method for prenatal screening of hereditary disorders has been developed; to this end, restriction Enzymes are used to map DNA extracted from embryonic Cells present in Amniotic Fluid.

Class="center">Table 7.3. Major serum enzymes used in clinical Diagnostics. Many of the listed enzymes are not specific to the diseases indicated in the table; additional data on the conditions under which The activity of these enzymes changes are given in the Appendix

In principle, DNA probes can be constructed for the diagnosis of the majority of Genetic Disorders. For example, for the prenatal detection of thalassemia (a defect in the synthesis of Hemoglobin subunits; see Ch. 6), a DNA probe can be synthesized from a fragment of the Gene encoding the normal hemoglobin subunit and used to detect the shortening or absence of a restriction fragment caused by a deletion in this gene. Such deletions are characteristic of certain types of a-thalassemia and several rare types of ß- and ß, δ-thalassemia (Dozy, Forman, Abuelo, 1979; Kan, Chang, Dozy, 1982). An alternative approach has also been proposed, based on the Construction of a synthetic cDNA that hybridizes with a ß-globin sequence containing a nonsense mutation characteristic of certain types of ß-thalassemia (Pirastu et al., 1984). The absence in plasma of a, -antitrypsin, a protease inhibitor, leads to The Development of emphysema and early liver cirrhosis. Inactive a,-antitrypsin was detected using a DNA probe constructed on The basis of the inactive mutant allele of the a, -antitrypsin gene (Kidd et al., 1983).

Hybridization probes can also be used to detect genetic alterations that lead to the loss of a restriction site (see Ch. 38). For example, Sickle Cell anemia is characterized by a point mutation in the GAG codon (Glu), resulting in the appearance of a GTG codon (Val); this mutation in the ß-globin gene can be detected by analyzing as little as 10 ml of amniotic fluid and using the restriction endonuclease Mst II or Sau I (Orkin et al., 1982).

DNA probes can also be used to detect DNA sequences that are tightly linked to the gene of interest but do not belong to the gene itself. Such analysis can be applied to detect chromosomal polymorphism (differences in the sequences of homologous Chromosomes). Cleavage of DNA with a restriction endonuclease in such cases yields distinct restriction maps (sets of DNA fragments), indicating differences in the base sequences of homologous genes. This phenomenon is termed restriction fragment length polymorphism (RFLP). Restriction analysis reveals two hybridization bands (if the genes were identical, only one band would be observed). Offspring who have inherited the defective chromosome also exhibit a single hybridization band, but its position differs from that of normal chromosomes. The RFLP phenomenon has been used to detect the gene for sickle cell anemia (linked to the Hpa I restriction site) as well as ß-thalassemia (linked to the Hind III and Bam HI restriction sites) (Little et al., 1980; Woo et al., 1983).

Screening based on restriction fragment polymorphism has also been used for the early diagnosis of phenylketonuria (Woo et al., 1983) (see Ch. 31). It should be noted, however, that the gene responsible for phenylketonuria itself does not affect the distribution of restriction fragments, but it is tightly linked to a restriction polymorphism site. The Prospects for Using restriction fragment polymorphism may be associated with the search for disease-causing genes linked to a polymorphic region. This approach has already been applied to screen for Mutations leading to the development of retinoblastoma tumors (Cavenee et al., 1983) and Huntington's disease (Gusella et al., 1984).

Further Examples of the diagnostic use of restriction fragments will be given in Ch. 36.

References

General Enzymology

Boyer P. D., Lardy H., Myrbäck K. (eds.) The Enzymes, 3rd ed., 7 vols. Academic Press, 1970—1973.

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Nomenclature

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Enzyme Assay and Purification

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