3. Direct diagnosis of sickle cell anemia using RFLP: The genetic disorders of Hb are the most common genetic diseases in humans. In the case of sickle cell anemia (Fig. 34.17), the point mutation that gives rise to the disease (see p. 35) is actually one and the same mutation that gives rise to the polymorphism. However, direct detection by RFLP of diseases that result from point mutations is limited to only a few genetic diseases.
a. Early diagnostic efforts: In the past, prenatal diagnosis of sickle cell anemia involved the determination of the amount and kinds of Hb synthesized in the nucleated red cells obtained from fetal blood.
However, the invasive procedures to obtain fetal blood have a high mortality rate (~5%), and analysis cannot be carried out until late in the second trimester of pregnancy when HbA (and its HbS variant) begins to be produced.
b. RFLP analysis: In sickle cell anemia, the sequence alteration caused by the point mutation abolishes the recognition site of the restriction endonuclease MstII: CCTNAGG (where N is any nucleotide; see Fig. 34.17). Thus, the A-to-T mutation in codon 6 of the βS-globin gene eliminates a cleavage site for the enzyme. Normal DNA digested with MstII yields a 1.15-kb fragment, whereas a 1.35-kb fragment is generated from the βS gene as a result of the loss of one MstII cleavage site. Diagnostic techniques that allow analysis of fetal DNA from amniotic cells or chorionic villus sampling rather than fetal blood have proved valuable because they provide safe, early detection of sickle cell anemia as well as other genetic diseases. [Note: Genetic disorders caused by insertions or deletions between two restriction sites, rather than by the creation or loss of cleavage sites, will also display RFLP.] 4. Indirect diagnosis of phenylketonuria using RFLP: The gene for phenylalanine hydroxylase (PAH), the enzyme deficient in phenylketonuria ([PKU] see p. 270), is located on chromosome 12. It spans ~90 kb of genomic DNA and contains 13 exons separated by introns (Fig. 34.18; see p. 442 for a description of exons and introns). Mutations in the PAH gene usually do not directly affect any restriction endonuclease recognition site. To establish a diagnostic protocol for PKU, DNA from family members of the affected individual must be analyzed. The goal is to identify genetic markers (RFLP) that are tightly linked to the disease trait. Once these markers are identified, RFLP analysis can be used to carry out prenatal diagnosis.
a.
Mutant gene identification: Determining the presence of the mutant gene by identifying the polymorphism marker can be done if two conditions are satisfied. First, if the polymorphism is closely linked to a disease-producing mutation, the defective gene can be traced by detection of the RFLP. For example, if DNA from a family carrying a disease-causing gene is examined by restriction enzyme cleavage and Southern blotting, it is sometimes possible to find an RFLP that is consistently associated with that gene (that is, they show close linkage and are coinherited). It is then possible to trace the inheritance of the gene within a family without knowledge of the nature of the genetic defect or its precise location in the genome. [Note: The polymorphism may be known from the study of other families with the disorder or may be discovered to be unique in the family under investigation.] Second, for autosomal-recessive disorders, such as PKU, the presence of an affected individual in the family would aid in the diagnosis. This individual would have the mutation present on both chromosomes, allowing identification of the RFLP associated with the genetic disorder.
b.