Hereditary Haemochromatosis

Introduction:

Genetic haemochromatosis is an autosomal recessive disorder that causes the body to absorb and store too much iron. This overload of iron builds up in organs and causes organ failure. The HFE gene is located on chromosome 6 and is the gene responsible for the regulation of iron absorption in the body. Haemochromatosis is most commonly associated with one of two common mutations within the HFE gene, these are the C282Y and H63D mutations.

Detection of these genotype mutations is achieved by the use of the restriction enzymes RsaI and MboI. RsaI recognizes the DNA sequence GTAC. The C282Y mutation alters the DNA sequence from GTGC (wildtype) to the GTAC sequence which is recognized and subsequently cut by RsaI. The restriction fragments may then be separated by agarose gel electrophoresis. MboI recognizes the sequence GATC. The H63D mutation of the wildtype sequence (GATG) results in this sequence and therefore individuals possessing this mutation can be identified using MboI restriction enzyme and agarose gel electrophoresis.

Methods:

To conduct the restriction enzyme digest and agarose gel electrophoresis, it was necessary to first amplify the DNA from our original sample so that there was enough to work with in the further stages of analysis. This was conducted using a Polymerase Chain Reaction or PCR. We prepared two master mixes which where to be used to conduct the PCR. Each master mix contained one of two different sets of primers (forward and reverse), specific to the regions where the H63D and C282Y mutations occur. It was essential that the correct mastermix was paired with the DNA samples, so that the 282 controls were amplified at this region rather than the 63 region and vice versa. It was also important for the unknown samples to undergo PCR with each of the primer sets, because being wild type for one of the mutations does not mean that individual will be wild type for the other mutation and they could still possess the predisposition to haemochromatosis.

Next the restriction enzyme digestion was conducted by adding either RsaI or MboI into the samples depending on which region they were amplified at. Each sample (containing an added loading dye) was loaded into a separate well on an agarose gel and an electric current was run through the gel to separate any fragments resulting from the digestion on the basis of their size. The smaller fragments travel further down the gel and larger fragments remain near the wells in which they were first placed. A "ladder" was also included in the gel. This is a sample that has been digested into fragments of known size to act as a guide to the size of the fragments that result from the restriction digest of our control and unknown samples. The fragments were visualized on the gel by exposing it to UV light on a trans-illuminator and photographing the gel.