The Molecular Analysis of a Human Mutation

How does one proceed when one has a patient with a genetic disorder known or suspected to be due to a mutation in a particular gene? For example, consider a patient with a diagnosis of β-thalassemia, an autosomal recessive defect in the β-globin gene. The initial diagnosis is generally made on the basis of clinical and hematological findings alone. However, it is important to examine the gene itself, first to confirm the clinical diagnosis and second, to determine the specific mutation in the β-globin locus both for future use in carrier testing and possible prenatal diagnosis in the patient's family, and for increasing the understanding of the relationship between specific mutations in the gene and the resulting patho-psychology.Several tests can be used initially to examine the gross integrity of the β-globin gene itself and its mRNA. Is the gene present in the patient in both normal amount (i.e., two copies) and structure? Or is one or both copies of the gene deleted or structurally rearranged, as has been described in some cases of β-thalassemia? If the gene is present, is it transcribed? The Southern blotting technique is now a standard method for using a cloned gene probe (in this case, for the β-globin gene) to examine the integrity of a DNA sample. Southern blotting can address the question of whether it is grossly normal in structure. By this method one can detect large molecular defects that are well below the level of sensitivity of chromosome analysis. However, as currently used in diagnostic laboratories, it cannot reveal the presence of single mutations, such as base-pair changes or very small deletions of only a few base pairs. In order to examine whether mRNA is present, a technique called Northern blotting is used. This approach also enables one to detect major changes in mRNA levels or structure for a specific gene, but not to detect minor alterations.Having asked whether there are gross changes in the gene or in its mRNA, one can proceed to a number of methods developed to examine gene structure and expressions at increasingly finer levels of analysis. In β-thalassemia, as in many other genetic disorders, particular mutations responsible for the disease in other patients. To determine whether one of these already known mutations is responsible for a particular case of β-thalassemia, one can apply particular direct molecular tests. Some of these entail the approach of allele-specific oligonucleotides (ASOs) that enable one to detect specific single base-pair mutations. In addition, it may be desirable to actually clone the mutant β-globin genes (or cDNAs) from the patient for comparison with a normal β-globin gene. Cloning of individual mutant genes (or portions of genes) from a patient's material is facilitated by use of polymerase chain reaction (PCR) to specifically generate many million of copies of a particular gene fragment. Once the mutant gene is isolated, one can then analyze it at the finest level possible by determining the DNA sequence of base pairs in the mutant gene for comparison with the normal gene. In this way, the specific mutation responsible for the genetic disorder in the patient can be determined and used to develop direct screening tests for that mutation in the patient's family.