Imagine you were a member of a family that has a history of HNPCC. Would you want to know if you have inherited the mutation? Knowing whether you are at risk for colon cancer could make a huge impact on your life. People with known HNPCC start colon screening around age 20 instead of their 40’s or 50’s as in the general population. Through this screening, any polyps that are developed can be caught and removed early, before they have the chance to turn cancerous.

So how do laboratories test for mutations in the MSH2, MSH6, and MLH1? One way would be through sequence analysis, where the DNA at those three critical gene locations is sequenced and any changes are detected between the patient’s DNA sequence and a normal/standard sequence. This method is very accurate but can also be very costly. This is the method typically used for clinical testing in the U.S. A blood sample is drawn from the patient and it is sent to a testing laboratory that specializes in this testing technique. The DNA is extracted from the patient’s blood and the sequence analysis is performed.

There are other techniques that can also be utilized to check for mutations in the DNA without actually sequencing the DNA. These other detection methods take advantage of the functional changes caused by the DNA mutation. For example, a change in the DNA sequence could cause an altered protein or enzyme product. There are tests that can be performed to see if a specific protein is being produced or if an enzyme is active. The results from these tests can demonstrate underlying genetic changes, although they are actually biochemical tests. They are also performed by drawing blood from the patient, but instead of extracting the DNA to the analyzed the serum is tested for the protein/enzyme.

A DNA mutation can also cause a change in something called a restriction enzyme target sequence. Restriction enzymes and their function are explained in detail below, but they essentially cut the DNA at specific target sequences. A mutation in the DNA can create a target sequence or could destroy one that is normally present. Based on whether the restriction enzyme cuts the DNA can determine whether a person has a specific mutation. The key to these other detection methods is that for them to be productive, the laboratory has to be looking for a very specific mutation. For many genetic disorders, including these cancer syndromes, there is not one single mutation in every positive patient. In these cases the best detection method is sequence analysis. However, if a mutation is identified in a family and it is known that it causes on of these functional changes, then these other detection methods could be employed.

In the family discussed in this laboratory exercise, there is a known HNPCC mutation running in the family. It is also known that this mutation is a single nucleotide change in the DNA that causes a new target site for a restriction endonuclease. To test members of this family for the mutation, the restriction enzyme can be applied to their DNA. If the enzyme cuts the DNA at the specific point, the mutation is present. Read below to find out how this testing is done.