Molecular Diagnostics
Genomic Evaluation of Risk of Atrial Fibrillation
and Alzheimer’s Disease
Session You Attended
Abstract
The aim to of this series of experiments is to examine a patient’s DNA for association to atrial fibrillation (AF) and Alzheimer’s disease. The single nucleotide polymorphism (SNP) rs2200733, associated with an increased chance of development of atrial fibrillation. Two SNPs: rs439358 and the rs7412, located within exon4 of the human apolipoprotein E (APOE) gene are associated with increased risk of developing Alzheimer’s disease. Isolation of the DNA from patient white blood cells, followed by PCR-RFLP and cycle sequencing are among the techniques utilized. The patient was found to the CT genotype at rs2200733 so an increased risk for AF, while the experiment was inconclusive for the patent’s risk for Alzheimer’s disease.
Introduction
Small changes in some locations of the human deoxyribonucleic acid (DNA) sequence can result in an outward difference in the organism, that is, a phenotype that can be observed or measured (Griffiths et al., 2000). Phenotypes can be the absolute expression of a particular trait or even just an increased tendency to exhibit a particular trait. One phenotype that has been shown to be due to small changes in the DNA of humans is an inherited increased tendency to exhibit atrial fibrillation (Mohanty et al., 2013). Atrial fibrillation is rapid heart beat that can cause heart attacks and early death. (Bond University, 2013). A second is the inherited increased tendency to develop Alzheimer’s disease (Nickerson et al., 2000). Alzheimer’s disease is a neurological disease that negatively affects memory and brain function (Bond University, 2013).
This experiment involves looking for single nucleotide polymorphisms (SNPs) in the DNA isolated from a patient sample to determine the patient’s genotype. The SNPs linked with these two disease tendencies are not absolute, that is, if the changes are observed in the DNA sequence it only means that the patient has an increased chance of developing the clinical problem, not the absolute certainty (Mohanty et al., 2013; Nickerson et al., 2000). Nevertheless, this information can be useful to a patient from an awareness point of view therefore motivating the patient to make lifestyle changes (Bitzer, 2010). The aim of this experiment is therefore determining the genotype of the patient at two different specific DNA locations that may have implications for Alzheimer’s disease and susceptibility for AF.
Methods
Methods were as described in the Laboratory Manual (Bond University, 2013).
As described in the cited methods, the DNA was isolated using a Qiagen test kit.
Results
The experiment began with the isolation of genomic DNA from white blood cells (WBC) within a whole blood sample. After extraction, we determined the amount of DNA isolated and its purity using spectrophotometry. (Q1.3) A spectrophotometer works by collecting light, dividing the light into the components of the spectrum, producing a digital signal that describes the amount of light in the selected spectral component, then reading and displaying that signal out using a computer (BW TEK, n.d.). The results of these measurements are recorded in Table 1.1.
WBC count/mL X amount pg/cell X 1 μg /106 pg X starting volume μL X
1 mL/103 μL = μg Theoretical Yield
5.3 X 106 cells/mL X 6.6 pg/cell X 1μg/106 pg X 350 μL X 1mL/103μL = 12.24 = 12 μg Theoretical Yield
Actual Yield/Theoretical Yield X 100 = Percentage Yield
5.1 μg /12μg = .425 X 100 = 43% Percentage Yield
In particular, the concentration of DNA found was figured by multiplying the A260 reading by the double-stranded DNA conversion factor of 50μg/mL. As recorded in Table 1, this resulted in a concentration of 51μg/mL or 51ng/μL. As there was 100 uL of volume, the yield was 5100 ng/μL. The purity ratio (260/280) was 1.85, and contamination ratio (260/230) was 1.29.
After amplification of the gene desert region including rs2200733 SNP by polymerase chain reaction (PCR) samples of the amplified section were subjected to cleavage by the BclI restriction endonuclease. The rs2200733 SNP (C>T) results in the loss of BclI site in the amplified region so presence of the mutation results in a restriction fragment length polymorphism (RFLP). Digested and undigested samples were run on an agarose gel and the results are in the xid-532450.jpg file (attached as Figure I).
As seen in Figure 1, Lanes 1 and 11 are 100bp DNA ladders. Lanes 2, 6, and 9 have three bands. Lanes 3, 6, and 11 are undigested control DNA and lanes 2, 4, and 8 are undigested DNA from the patient, although lane 8 is very faint due to low DNA concentration. All of these lanes have one band.
The next part of the experiment involved amplifying exon4 of the APOE gene and subjecting this amplicon to sequencing to determine the presence or absence of the rs439358 SNP and the rs7412 SNP within that region. (Q1.7) The original primers used were tailed with M13 sequences because this would add the M13 sequences to the amplified portion. The added M13 sequences will anneal with the M13 primer available in the sequencing reaction. (Q1.8) Only one primer is used because sequencing is performed using singled-stranded DNA. By using only one primer, only one strand of the DNA is amplified.
Sequencing of the sample was unsuccessful in determining the identity of nucleotide 251 from the primer (indicated by N in that position in the sequence electropherogram, attached as Figure 2) and in the reference and patient sequence comparison (attached as Figure 3). The rs7412 position (nucleotide 389 from the primer) was found to be cytosine (C) (see Figures 2 and 3).
Discussion
The aim of this experimental series was to determine the genotype of a patient at locations within the DNA known to have small changes that are associated with the tendency to develop atrial fibrillation (SNP rs2200733, Mohanty et al., 2013) and Alzheimer’s disease (the combination of SNPs rs429358 and rs7412, Nickerson et al., 2000). The DNA was isolated at a yield of 43%. This is a low yield, especially for a commercial prep kit, so there likely were problems in the way the test kit was used or a very slight possibility that the kit itself was defective.
The DNA was found to be of good purity with a 260A/280A ratio value of 1.85, but as the contamination (260A/230A) ratio outside the expected ratio of 2.0 to 2.4, so there may be contamination with extraction chemicals (Bond University, 2013). This is further indication of problems with the kit. (Q1.2) Additionally, the DNA concentration found may be an overestimation if the amino acids in the DNA sequence have less than average amount of aromatic side chains (reduces absorption at 280 nm). The pH of the solution can also affect absorption values causing an overestimation if alkaline (Oxford Gene Technology, 2011).
The experiments continued with THE determine AT the SNP location associated with atrial fibrillation (rs2200733) using PCR-RFLP analysis with the BclI restriction endonuclease. After digestion, fragments were run on an agarose gel (Figure 1). PCR-RFLP is a good test for this SNP as the mutation results in the loss of a restriction endonuclease recognition site. (Q1.4) If the SNP is present, the restriction fragments size would be about 259bp as the mutation removes the BclI cleavage site, so no cleavage would occur and the amplicon would stay whole.
(Q1.5) The Bcl1 restriction size fragments seen with a TT genotype would be only the 259bp amplicon. With a CT genotype there would be the 259bp amplicon and the two fragments of 157bp and 102bp. With the CC genotype there would be only the 157bp and 102bp fragments. (Q1.5, p.14) The undigested PCR amplicon has only a 259bp fragment. The digested amplicon has the 259bp full length amplicon and the 157bp and 102bp fragments. (Q1.6) The rs2200733 SNP genotype of the patient was found to be TC. So we have determined that the patient is at higher risk for atrial fibrillation than someone without that mutation (Mohanty et al., 2013).
Finally, we determined the DNA sequence of the patient at the SNP locations associated with an increased risk of Alzheimer’s disease using amplification and sequencing. Unfortunately, a technical difficulty meant that nucleotide at the SNP rs429358 could not be determined. But the sequence located at SNP rs7412 was shown to be C. The phenotype results from a combination of these two SNPs (Bond University, 2013, 16).
(Q1.9) Specifically, the APOE genotype of our patient is either ε3 or ε4. The nucleotide responsible for rs429358 was not determined (“N”). However, one variant can be eliminated because the nucleotide at rs7412 was determined to be C, so the patient cannot be ε2. The results mean that the patient could have a normal or neutral phenotype, ε3 or one that confers an increased risk of Alzheimer’s, ε4 (Nickerson et al., 2000). Thus, with these possible results, redoing the sequencing is the best solution. The limitations of this experiment are with the quality of the DNA which could be solved with a different extraction technology other than the QIAmp kit by QIAGEN. A second possible limitation is the accuracy of the sequencing. (Q1.10) The most common way to improve accuracy is to run the sequence a second time and check for differences. A different method of sequencing could also be used, such as the method promoted by Pacific Biosciences, that uses two runs of the same sequence, one in a single pass machine and one in a second generation machine, and a software program to reconcile differences (Strickland, 2012).
The most relevant conclusions from this experiment is that the patient does have an increased chance of developing atrial fibrillation and the patient may or may not have an increased change of Alzheimer’s. To be sure the test needs to be run again, perhaps using different technology such as the Pacific Biosciences method discussed in the paragraph immediately before this one.
References
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Bond University, 2013. Molecular Diagnostics Laboratory Manual. [pdf] Robina, Queensland, AU:Author.
BW TEK, n.d. How does a spectrometer work? [online] Available at: <http://bwtek.com/spectrometer-introduction/> [Accessed 24 June 2013].
Griffiths, A., Miller, J., and Suzuki, D. et al., 2000. How DNA changes phenotype. In An introduction to genetic analysis. (7th ed) [online]. New York, NY: W.H. Freeman. Available at: <http://www.ncbi.nlm.nih.gov/books/NBK21955/> [Accessed 25 June 2013].
Mohanty, S., Sangtangeli, P., and Bai, R. et al., 2013. Variant rs2200733 on chromosome 4q25 confers increased risk of atrial fibrillation. Journal of Cardiovascular Electrophysiology. 24(2), 155-61.
NCBI, n.d., Restriction fragment length polhymorphism. [online] Available at: <http://www.ncbi.nlm.nih.gov/projects/genome/probe/doc/TechRFLP.shtml>
[Accessed 25 June 2013].
Nickerson, D., Taylor, S., Fullerton, S., et al., 2000. Sequence diversity and large-scale typing of SNPs in the human apolipoprotein E gene. Genome Research. 10(10), 1532-45.
Oxford Gene Technology, 2011. Understanding and measuring variations in DNA sample quality, [online] Available at: <http://www.ogt.co.uk/resources/literature/483_understanding_and_measuring_
variations_in_dna_sample_quality>[Accessed 24 June 2013].
Strickland, E., 2012. 99.9 percent accurate genome sequencing. IEEE Spectrum. [online] 2 July. Accessed at: <http://spectrum.ieee.org/tech-talk/biomedical/diagnostics/99-9-percent-accurate-genome-sequencing> [Accessed 24 June 2013].
