Haemochromatosis

HAEMOCHROMATOSIS DETECTION USING PCR-RFLP

INTRODUCTION

Hereditary Haemochromatosis (HH), first described in 1865, is a genetic disorder of metabolism, characterized by progressive iron overload resulting from abnormalities in intestinal iron absorption and or release of iron from reticuloendothelial cells . It is an autosomal recessive disorder, where the body accumulates excessive iron, which is deposited in a variety of organs. Iron cannot be excreted, thus, the excess builds to toxic levels in tissues of major organs such as the liver, heart, pituitary, thyroid, pancreas, lungs, and synovium (joints). These organs cease to function adequately and eventually become diseased. Serious illnesses such as diabetes, cirrhosis, hepatoma, hypogonadotrophic hypogonadism, cardiomyopathy and arthritis may be a consequence of this disease . It affects one in every three hundred Caucasians, and one in nine is a carrier , hence, making its early detection vital. The gene responsible for HH (HLA-H) was recently identified on the short arm of chromosome 6 and is thought to be mainly caused by a mutation of a gene called HFE, which allows excess iron to be absorbed from the diet . This mutation is known as C282Y. A single point mutation occurs, in which the amino acid cysteine at position 282 changes to a tyrosine . To develop haemochromatosis two genes, one from each parent, are required to be C282Y. However, not everyone with the mutation may develop the disease and it may occur if only one C282Y gene is present (4). 77.5% of affected individuals have two copies of the C282Y mutation, one inherited from each parent, while about 4% have a single copy of the mutation and one normal HFE gene .

First proposed in early 1970’s, Polymerase Chain Reaction (PCR) has been identified as a simple, robust, speedy, and most of all, flexible method that can be used to detect haemochromatosis . In this technique, specific DNA sequences are amplified for the detection of mutations that may be present, allowing early diagnosis of hereditary heamochromatosis (see figure 1). It is a major development in the analysis of DNA and RNA. The requirements of the reaction are simple, consisting of deoxynucleotides to provide both the energy and nucleosides for the synthesis of DNA, template, primer, DNA polymerase, and buffer containing magnesium . The crux of the PCR procedure involves three steps, including denaturation at high temperatures, annealing of primers, and extension, which are repeated for 30-40 cycles.

The aim of this experiment was to use a PCR-RFLP to determine the presence of HLA-H gene responsible for hereditary haemochromatosis. The genomic DNA containing the Cys282Tyr mutation was amplified by PCR and the mutation was detected in several individuals. In addition, it is also known that this mutation results in the establishment of a new RsaI site into the DNA, which was identified by restriction digestion of the PCR product . Moreover, vital variables, such as magnesium ion concentration, primer annealing temperature and template concentration, were also examined to establish their relationship with the efficiency and specificity of target DNA by the PCR.

METHODS

The procedures dictated in ‘Biochemistry Genes and Disease’, Practical manual. 2004 pages 21-27 were followed. The only amendment that was in experiment one. The step “Overlay reactions with 100ul mineral oil” was excluded.

RESULTS

Figure 1 The PCR product obtained from the amplification of HLA-H Genomic DNA. The following could be seen from the electrophoresis analysis:

· Lane 1 depicts bands for pUC19/HpaII markers.

· Lanes 2 & 3 display the heterozygous and homozygous bands (alleles) respectively.

· Lane 4-10 show bands for ‘Wild Type’, which represents two normal alleles, thus not being exposed to the HH disorder.

· Lanes 5-7 depict the effects of varying magnesium concentration, as tested in experiment 1. At 0ul, there is no band present which can be seen in Lane 5. However, as magnesium is added, in Lanes 6 and 7, bands appear at varying intensity. At 1ul an ambiguous, band can be seen, which is smudged and undefined in nature, as seen in Lane 6. In Lane 7, at 2ul, a bright, defined band is visible.

· Lanes 8-10 depict the results obtained as a consequence of altering the template concentration, as dictated in experiment 2. At 1ul in Lane 8 results illustrates faint, unclear