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 band. However, its clarity is seen to improve and seen being optimal in Lane 9, at 3ul of the template. Additional DNA in Lane 10, at 10ul, shows a compromise in the quality of the band. The band appears to be bright, yet inadequate in thickness.





Figure 2 Electrophoresis analysis of Wild Type using annealing Temperature Dependence. This illustrates the results obtained for experiment 3, in which the effects of temperature on PCR were observed:
·     Lane 1 depicts bands for pUC19/HpaII markers.
·     Lane 2 illustrates the presence of unclear, clustered bands, lacking any definition, when treated at 45C.


















Figure 3 Gel electrophoresis analysis of RsaI digestion of HLA-H PCR product. This figure depicts electrophoresis of Heterozygous and Homozygous alleles compared to standard markers.

·     Lane 1 depicts bands for pUC19/HpaII markers.
·     Lane 2 displays two bands at 260bp and 135bp, which represent the two normal alleles of the ‘Wild type’.
·     Lane 3 illustrates the presence of 260bp, 135bp, 105bp and 30bp bands, which exemplifies the presence of one normal and one mutated allele, proving it to be heterozygous in nature.
·     Lane 4 shows bands at 260bp, 105bp and 30 bp. This depicts the presence of two mutated alleles, and fulfills the requirements for being homozygous in nature.

Figure 4 The class PCR product obtained from the amplification of HLA-H Genomic DNA.
















Table 1a & b. Final concentrations of each reagent in the PCR reaction.

           REAGENT     
     dNTPs     Tris buffer     Primers
CONCENTRATION     0.44mM     20.02mM     0.001012mM



Table 1b
                 REAGENT           
     Mg2+ (vol=1.0ul)     Mg2+ (vol=2ul)     DNA (1ul)     DNA (3ul)     DNA (10ul)
CONCENTRATION     2.0mM     4.0mM     0.8ng     2.4ng     8ng

Table 2 Comparison of base pairs to mobility for pUC19/HpaII

Mobility (bp)     Marker size (mm)
7     331
11     242
13     190
17     147
22     111
32     67

The distance migration along the side was measured using a ruler to assist in constructing a calibration curve to determine the sizes of the DNA fragments produced by PCR. See Figure 5 for the graph. A mobility V’s log Marker size was plotted to gain this.

DISCUSSION
Affecting 1 in 200 Australians of Anglo-Celtic descent , Hereditary Heamochromatosis (HH) is one of the most common genetic disorders with high morbidity and mortality which is preventable if diagnosed early .

In 1996, Feder et al. drew a connection between mutations in a haemochromatosis gene and hereditary haemochromatosis. The HFE gene encodes a protein similar in structure to the MHC class 1 molecules. The protein encoded by the HFE gene interacts with the transferrine receptor and is involved in the regulation of iron absorption. He found two point mutations which could repeatedly be found in patients with hereditary haemochromatosis. The replacement of G (guanine) by A (adenine) at position 845 in the HFE gene causes in the protein a change from cysteine (C) to tyrosine (Y) at the amino acid position 282 (mutation C282Y). This mutation occurs heterozygously in approx. 5% of the total population (6). Over 90% of all patients with haemochromatosis carry the allele C282Y homozygously . Following this characterisation of the HFE gene in 1996, genetic testing for hereditary haemochromatosis has become available. Studies indicate that susceptibility to hereditary haemochromatosis have risen from 14 414 in 1999 to almost 30 000 in 2002 . Many symptoms are reversible, and normal lifespan is possible. Therapy after late detection is less effective, particularly if cirrhosis is present, and prognosis is guarded (11). These implications and prevalence thus impose the need for the early detection and treatment for HH .

Until the recent development of a DNA test, early diagnosis has been difficult. Despite the high prevalence of HH, most cases remain undiagnosed. Testing has been based upon measurements of serum iron, iron binding capacity, transferrin saturation, and ferritin concentration. However, these tests are imperfect, without clear "cut-offs" for results indicating affected status. Liver biopsy has traditionally been used for definitive diagnosis, but this invasive procedure is performed late in the course of the disorder. A simple, non-invasive and inexpensive DNA test known as PCR (Polymerase Chain Reaction) analyses of targeted gene regions has been identified as a diagnostic method for the detection of HH (11). As mentioned, this technique is a primer extension reaction for amplifying specific nucleic acids in vitro. It allow a short stretch of DNA (usually fewer than 3000 bp) to be amplified to about a million fold so that one can determine its size, nucleotide sequence, etc . It is comprised of three major components, including Denaturation (94°C), Annealing (54°C), and extension (72°C), which are repeated for 30 or 40 cycles . It allows the amplification of genomic DNA containing the Cys282Tyr mutation and the identification of the mutation. Restriction digestion of the PCR product allows the detection of the new RsaI site on the DNA, which is a categorist establishment of HH disease. The specificity and efficiency of the amplification of target DNA revolves around certain parameters that affect PCR (7), including components such as presence of magnesium, primer annealing temperature, and template concentration.

‘All thermostable DNA polymerases require free divalent cations’ (7) usually Mg2+ for activity. dNTP– Mg2+ complexes interact with the
sugar-phosphate backbone of nucleic acids and influence the activity of Taq
DNA polymerase. Hence, altering the concentration of MgCl2 can lead to one
Primer or template pair behaving significantly differently from another under
Identical conditions (18). A study corroborated this phenomenon, by suggesting that ‘Mg++ is known to play a critical role in amplification as it can affect DNA strand denaturation, primer annealing specificity and enzyme fidelity ’. dNTPs and oligonucleotides bind Mg2+, thus the molar concentration of the cation must exceed the molar concentration of phosphate groups contributed by dNTPs and primers. An imprecise magnesium concentration will reduces and in some instance, hinders the amplification of PCR product entirely. This was challenged in the experiment, in which the PCR reactions were observed in light of varying magnesium concentration. Electropherosis analysis in Figure 1 endorsed the requirement of having an adequate magnesium concentration to obtain optimal PCR results. It depicts the varying samples of PCR that were electrophoresed on 4% high resolution agrose gels for three varying Mg2+ concentrations. At 0ul in lane 5, no band is visible, thus signifying a lack of PCR product being formed. At 1ul, in lane 6, a band can be seen, however, is ambiguous, smudged, distorted and undefined in nature. This exemplifies scarcity in the Mg2+ concentration, which hinders the adequate formation of the PCR product. At 2ul, in lane 7, a bright defined band is present, indicating that this concentration is optimal for gaining the PCR product. It could thus be concluded, that as the concentration of Mg2+ was increased, more product was obtainable. However, its absence led to an absence of the product, hence making its presence vital for the PCR reaction to work.

Another variable that was challenged in this experiment was the primer annealing temperature. It has been found that the success of a PCR depends immensely on the specificity with which a primer anneals only to its target, and not non-target sequence, thus, making it imperative to optimize this molecular interaction. Whether a primer can anneal only to its complement or also to sequences that have one or more mismatches to the primer depends critically upon the annealing temperature . Generally, it follows a proportional relationship, as the higher the annealing temperature the more specific annealing of the primer to its perfect matched template and thus, the greater the likelihood of only target sequence amplification. On the contrary, the lower the temperature, the higher the presence of mismatches between template and primer, resulting in increased amplification of non-target sequences (18). Another source corroborated this, however further stated that ‘if the annealing temperature is too high, the oligonucleotide primers anneal poorly, if at all, to the template, and the yield of amplified DNA is very low’ (7). On the contrary, employing a temperature, which is below a critical level will result in ‘nonspecific annealing of primers’, causing ‘an amplification of unwanted segments of DNA’ (7), thus making the use of appropriate temperature for the annealing step critical and imperative. This phenomenon was challenged, in this experiment. As illustrated in Figure 2 when 45°C was used as the annealing temperature, clusters of smears were present, which lacked clarity or definition. This exemplifies the amplification of undesired segments of DNA, resulting in the lack of specificity, thus proving this annealing temperature inadequate for optimal results. However, class results in Figure 4, Lanes 6 and 7 depict the effects of using 62°C as the annealing temperature. Theoretically, Lane 6 should lack the presence of any band appearing, as the tube that was tested in this Lane, lacked any template DNA. This was observed in the gel electophoreses in Figure 4, as only the tube with the DNA (Lane 7), gave a bright, defined band. Lanes 8 and 9 show the treatment under 45°C, with Lane 8 lacking DNA, thus showing no band. A band was visible in Lane 9, however, its size was inadequate, as it was too thick, again, supporting the findings in Figure 2, that 45°C was not an adequate temperature for optimal results. Thus it can be verified that the usage of 62°C gave optimal results, with specific annealing of the primer to its matched template.

The amount of DNA template strongly influences the outcome of the PCR reaction. This association was evident in the results, in Figure 1, in Lanes 8-10, which showed the effect of varying the template concentration on the reaction. At 1ul in Lane 8, a faint, distorted band was visible. It lacked clarity and form. However, as the template concentration was increased to 3ul in Lane 9, a clear, defined band was seen to illuminate, suggesting the optimal concentration for the template. When the concentration was increased further to 10ul in Lane 10, there appeared to be a compromise in the quality of the band. The band appeared to be bright, yet inadequate in thickness. Hence, these findings showed that 3ul was the optimal template concentration to achieve adequate results. Any concentration lower than this compromised the clarity and visibility of the product, whilst increasing the concertration above this level, contrarily resulted in emergence of smudging and inadequate thickness of the product.

As discussed, haemochromatosis is an autosomal recessive disease caused by mutations in the HFE gene. To develop haemochromatosis an inheritance of two defective gene (homozygous) from each parent is required to be C282Y. However, a person who inherits a defective gene from only one parent is heterozygous, and is a carrier but does not develop the disease. It has been discovered that this mutation results in the establishment of a new RsaI site into the DNA. In the experiment, this was identified by restriction digestion of the PCR product. The genomic region which was amplified was 400 base pairs. In unaffected individual (Wild Type), a single RsaI site exists, which gives 260base pairs and 135 base pairs bands when its cut. In an affected individual, an additional RsaI site eventuating in 260 base pairs, 105 base pairs and 30 base pairs bands (9). In accordance to these findings, in Figure 3 it can be determined that Lanes 3 and 4, denoting heterozygous and homozygous respectively contained the Cys282Tyr substitution. In Lane 3, the DNA is cut at 260bp, 105bp and 30bp. However, since this individual has only one abnormal allele, he will not show the disease, but will be a carrier fro HH. In Lane 4, there is an addition of an RsaI site, showing a cut at 260bp, 105bp and 30bp. This individual is seen to possess two abnormal alleles, suggesting, that he is homozygous, thus developing HH disease.













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