Genotyping ApoE Variants: For Early Diagnosis of ARC

Genotyping ApoE variants: Predictor of rare cancer in young adults

According to Yamashiro (2017), The rare ApoE related cancer (ARC) occurs in mostly in young adults with 80% of all cases being in people between the ages of 20-30 years old. ARC is unbiased to gender and there is evidence that it is a hereditary disease linked to the inheritance of eight variants of the gene Apolipoprotein E (ApoE), that are spread out through the gene, including two that are in intronic sequences. These alleles seem to be recessive with some of them that when they are recessive homozygotes being strong predictors of ARC. It was also sometimes found that when there is heterozygosity of two recessive ApoE alleles, one being ApoE4, can act as recessive homozygotes. However, ApoE4 has the weakest association with cancer at less than 2%, but when another ApoE allele is present the ApoE4 allele pairs as a normal ApoE gene (Yamashiro, 2017a).

The ApoE gene encodes for the apolipoprotein E which combines with fats in the body to form lipoproteins (US National Library of Medicine, 2017). The ApoE lipoproteins are responsible for maintaining normal cholesterol levels in the bloodstream and the brain by transporting cholesterol and other fats in the bloodstream and assisting deposition of amyloids “and the clearing of deposits from the parenchyma of the brain (Garg & Roth, 2015).” The allele ApoE4 has the weakest link to ARC, less than 2% and when it pairs with another recessive ApoE allele they seem to be equivalent to a normal ApoE gene (Yamashiro, 2017a). Some variants of the ApoE gene increase the risk of developing heart disease, Alzheimer’s diseases (AD), and ARC. Compared to the other alleles ApoE 4 increases the risk for AD and it was also found to be a risk factor cerebral amyloid angiopathy, dementia, and multiple sclerosis (Zhong, et al., 2016).

Tests for Genotyping ApoE Variants

Early detection of ApoE alleles that are high-risk factors for ARC is necessary for the most effective treatment of the cancer. It is essential to use rapid and cost-effective tests to genotype all 8 recessive alleles of ApoE to determine carrier status of the recessive alleles and homozygosity of the recessive alleles that will most likely lead to ARC development. There are several test methods for ApoE genotyping and this case study will focus on RT-PCR, Oligonucleotide Microarrays, and Next Generation Sequencing.

Real-time PCR (RT-PCR)

Conventional polymerase chain reaction (PCR) which was conceptualized by Kary Mullis in 1983 and it has the ability to amplify specific nucleic acid sequences exponentially in a short amount of time. PCR amplification is achieved through multiple cycles of denaturation, annealing, and extension in a thermocycler that controls the temperature for each cycle (Kuslick, Chul, & Yamashiro, 2008). In the denaturation step the reaction mix (which contains the DNA template that will be amplified, a pair of primers, Taq polymerase, the four building blocks of the DNA which are the deoxyribonucleotide triphosphates (dATP, dCTP, dGTP,and dTTP), a salt with Mg²⁺ (a divalent cation), and a buffer) is heated for some time to cause the double-stranded DNA to dissociate in preparation for hybridization of the primers onto the DNA template. During the annealing step, the temperature is brought down in order for the primers hybridize on each DNA strand. Finally, in the extension step, the thermostable DNA polymerase, the Taq polymerase synthesizes the DNA strands with the primers to make new complementary DNA strands (Kuslick et al, 2008). The amplicons, which are the amplified double stranded deoxyribonucleic acids, are visualized as bands by agarose gel electrophoresis. Conventional PCR is useful for DNA amplification; however, it has a time-consuming procedure and data analysis of the results as each marker needs to be investigated separately by PCR, thus taking a long time to get to a final diagnosis (Irshad et al, 2016).

Real-time PCR (RT-PCR) is a PCR technique that allows for visualization of the DNA while it is amplifying by the addition of a fluorescent primer/probe to the reaction mix and running the reaction under ultraviolet light with a video camera recording each cycle, and translating the data into an amplification curve (Valasek & Repa, 2005). RT-PCR has the capability of genotyping all eight ApoE gene variants through SNP (single nucleotide polymorphisms) genotyping using TaqMan technology by amplifying each variant in a separate tube using a forward and reverse primer specific for each target sequence of the alleles. TaqMan is an RT-PCR system from Roche and utilizes a primer/probe with a reporter dye and a quencher dye attached, for visualization and follows a similar protocol to that of conventional PCR. The difference is, however, when the Taq polymerase is extending the DNA it encounters the probe and a 5′-3′ exonuclease activity will cleave the probe which in turn untethers the reporter dye away from the quencher dye releasing the signal from the reporter dye and the signal is then measured by the equipment that the reaction is being conducted in (Zhong, et al., 2016). Some advantages of SNP TaqMan RT-PCR are that it is a closed reaction system which reduces the risk of contamination of amplicons, has very little labor in the protocol, and takes less than a day to get to a final diagnosis. A disadvantage is that requires an RT-PCR machine that can read data at real time rather than using a fluorescence reader used for conventional PCR (Geyer, Reisbig, & Hanson, 2012).

Non-technical Parameters.

When designed optimally the primers for RT-PCR can be very accurate with high specificity and sensitivity of the results. There are many companies other than Roche, like Thermo Fisher Scientific that offer a variety of TaqMan assay formats for real-time PCR such as singles, 96-well plates, 384 microfluidic cards, and openArray plates (Thermo Fisher Scientific, 2017). The cost per assay for TaqMan can be from around $3 and goes up to $350 (Science Exchange, 2017a).

Oligonucleotide Microarrays

DNA microarray technology was originally designed to measure the RNA transcriptional levels of genes in a genome. With this technology, it is now possible gene expression patterns for studying diseases, disease progression, detect single nucleotide polymorphisms (SNPs), and identification for drug targeting. Microarrays use single stranded DNA sequences as probes just like in PCR to form complementary hybrids with the target DNA sequences to measure the expression of multiple genes. Thousands of DNA probes for the target sequences are bound, synthesized, or spotted to a silicon chip wafer similar to those used for computer microchips.

There are two main types of DNA microarray chips methodologies and it depends on the type of probes that are to be spotted (Trevno, Faclciani, & Barrera-Saldaña, 2007). One type was developed by Affymetrix that is adapted from the manufacturing of semiconductors and synthesizes short single-stranded oligonucleotides, about 22 nucleotides in length, in situ onto the wafer (Trevno et al, 2007) (Yamashiro, 2017c). The second type uses reverse transcription of messenger RNA (mRNA) to get complementary DNA (cDNA) for the cloning of the double-stranded DNA gene sequence, and then amplification of the open reading frames using PCR. The cDNA are the probes bounds to the wafer. A limitation of the cDNA method is that there is an uneven melting temperature due to the differences in the CG- content of the large open reading frames or cDNA sequence probes. There is also non-specific hybridization from overlapped genes, related sequences, and variations in splicing. The oligonucleotide method is designed in such a way that overcomes that of the cDNA probes, by designing the oligonucleotide probes to be complementary to the target sequence and redundantly detect the target segments (Pastinen, et al., 2000) (Trevno, 2007).

The extracted nucleic acid sequences are labeled with fluorescent dyes and are hybridized onto the DNA array through incubation and afterward, non-specific hybrids are washed off. The fluorescent dyes are detected through a laser in a confocal scanner that excites them and then produces a digital image of the microarray. Special software is used to analyze the image that assigns a final reading of a value that is relative to concentration in each spot of the probe of the target sequence being measured. There are some microarray methods that are “competitive two-dye assays” that uses two types of fluorophore dyes, one for the target sequence and the other for the reference sample (Trevno, 2007). The microarray reading assigns a ratio of the two dyes equal to the amount of the target sequence to the reference sample. This method is suitable to for measuring a small number of genes (Trevno, 2007).

Frequently the oligonucleotide microarray method is used for large scale multiplex genotyping of multiple alleles, mutations, and single nucleotide polymorphisms (SNP), and it would be the method of choice for genotyping the ApoE alleles (Pastinen, et al., 2000). Chromosomal microarray is a type of oligonucleotide microarray, that is commonly used in clinical laboratories as a genetic test for analyses of genomic copy number, SNP, karyotyping for visualization and analyses of chromosomal rearrangements like gains and losses (Miller, et al., 2010). Each ApoE variant sequence would be identified using two to three oligonucleotides for the sense and antisense strands. The array would have data points for the sense and antisense primers for analyses in order to reduce the occurrence of false positives. A genotyping software would then identify the variant sequences in each patient tested (Schrijver, 2005).

A downside of microarrays is that for some genetic carrier screening, such as Cystic Fibrosis carriers, a second tier of testing is often required to prove carrier status. The second tier is usually a more “comprehensive test such as differential gradient gel electrophoresis or denaturing high-performance liquid chromatography, followed by direct DNA sequencing to characterize the mutations identified by scanning techniques (Schrijver, 2005).”

Non-technical Parameters

A con of oligonucleotide microarrays is that they can take from a week to a month to get to a diagnostic result, but they do, however, have 80 – 98% analytical sensitivity and specificity. A pro is that the test can cost anywhere from $25 to $800 per sample, but still more expensive that TaqMan (Science Exchange, 2017b).

Next Generation Sequencing

DNA sequencing is the gold standard when it comes to genetic tests, however, its high costs make it difficult for routine use (Schrijver, 2005). In recent years there have been advances in DNA sequencing through Next Generation technologies (NGS), as they afford higher throughput and speed. There are three common NGS platforms, which are Roche 454, Illumina, and AB SOLiD. They are similar in that they measure and analyze signals that are emitted through a second strand of DNA to sequence the DNA. The way the second DNA strand is generated is where these platforms differ. Template DNA is split into smaller pieces, amplified, and then attached on a surface before sequencing (Pabinger, et al., 2014).

In the Roche 454 platform, DNA sequence fragments are ligated onto oligonucleotide adapters on beads that go through emulsion PCR that amplifies the DNA to amplify the copy number of the DNA fragments. The beads are diluted, then a single bead is dropped into each microwell of PicoTiterPlate. Pyrosequencing is then conducted by adding enzymes for sequencing and triphosphate nucleotides bases that release pyrophosphates when the bases encounter complementary bases on the DNA sequence that are on the beads. This produces light that is recorded detected by a CCD camera that denotes the triphosphate nucleotide base type in the DNA sequence in each well. This method is error prone as it misidentifies the length of nucleotides with identical bases (homopolymers) (Hodkinson & Grice, 2015).

The Illumina approach is the most widely used NGS platform because of it allows a large amount of data to be generated, with a low error rate and is cost effective. This method avoids homopolymers by using a sequencing by synthesis method that uses reversible dye terminators with one nucleotide per sequencing cycle (Hodkinson & Grice, 2015). The dye terminators are washed over a flow cell that has the oligonucleotides immobilized on it and had been hybridized with the DNA fragments. After the dye terminator has attached, the unbound nucleotides are washed away and the flow cell is imaged. Since the dye terminator is reversible it can be washed away after each cycle to get the identity of the next base pair. Illumina sequences shorter fragments, about 35 – 100 base pairs and uses a special program that uses an algorithm to determine the sequence (Hodkinson & Grice, 2015) (Yamashiro, 2017d).

The AB SOLiD platform is similar to 454, in that it starts with emulsion PCR but uses a sequences-by-litigation approach (Hodkinson & Grice, 2015). The DNA libraries are sequenced by “by 8 base-probe ligation which contains ligation site (the first base), cleavage site (the fifth base) (Liu, et al., 2012).” Di-based probes that are fluorescently labeled with four dyes, ligate to the DNA sequence and produce a fluorescent signal that is recorded. The sequences are read in multiple cycles since at least the first two bases are read with high confidence. This redundancy of this method reduces its error rate (Liu, et al., 2012) (Yamashiro, 2017d).

Non-technical Parameters

To cut time and money anyone of the NGS platforms could be used to only analyze chromosome 19 since that is where the ApoE protein is located. Roche 454 is the most expensive of the three starting at $8000 per sample and Illumina the cheapest with tests starting at $35 per sample. An advantage of the NGS technologies is the amount of data it can generate like mapping parts of or the whole genome of the individual and can be more sensitive to detecting rare sequences among related sequences (Hurd & Nelson, 2009).

Results

The SNP TaqMan RT-PCR test method would be the system of choice for genotyping the ApoE alleles. It has the highest analytical sensitivity and specificity and the most cost efficient. Although it does not give as much information as the NGS platforms in terms of epigenetics and genome mapping, it does get the job done within a reasonable amount of time. Microarrays and NGS need specialized software to perform bioinformatic analysis of the results to get a final diagnosis (Liu, et al., 2012) (Miller, et al., 2010). Whereas with SNP TaqMan each reaction tube has specific forward and reverse primers for each ApoE allele and can be visualized in real time, making it the easiest to use and fastest to get to a result (Zhong, et al., 2016). Table 1 compares the different technologies explained earlier for genotyping methods. Illumina is the only other technology that can compare to SNP TaqMan RT-PCR in terms of sensitivity and specificity, but it takes a bit longer and the cost can easily go up to thousands of dollars (Science Exchange, 2017b).

Validation

In order to develop the TaqMan RT-PCR assay, primers for each ApoE allele are designed through a software through a software such as Beacon Designer 7. An analysis is then done using sequences submitted to a database like GenBank on the primers/probes sequences to evaluate their ability to anneal to the target variants, by means of a BLAST analysis (Geyer et al, 2012). The primer/probe sequences that annealed with a 100% specificity to the target variants only, are chosen and are labeled with a different reporter fluorophore dye (e.g. FAM, TET, HEX) at the 5′ end and a quencher dye (e.g. TAMRA) at the 3′ end (Geyer et al, 2012) (Kutyavin, et al., 2000). The probes are then ordered from a company that manufactures probes for TaqMan such as Bioresearch Technologies in Novato, CA (Qu, Wanner, & Christ, 2011).

The next steps would be to optimize the PCR assay by testing the parameters of the different components that get put into the master mix, the concentrations of MgCl₂, primers/probes, DNA template, dNTPs, Taq polymerase, and buffer concentration. These are tested using a thermocycler that is equipped for TaqMan PCR, where the temperatures and timing for each step in the cycle are also adjusted to get optimal annealing, hybridization, and amplification of the DNA. The C_t value (cycle threshold) which is the number of cycles in a run that crosses the threshold is determined. Anything above the threshold is a positive indicator that the allele being tested is present (Qu et al, 2011).

For validation of the ApoE TaqMan PCR assay, the results are compared to results from a DNA sequencing analysis. The ApoE fragments are amplified by PCR with the designed primer/probes and then the products purified and sequenced by a DNA sequencer like the ABI 3730XL DNA Sequencer by Applied Biosystems (Zhong, et al., 2016).

Discussion

Eight ApoE alleles are linked to ARC disease and it has been determined the TaqMan RT-PCR would be the best assay to test for these alleles. Screening for ARC related alleles before cancer develops is very beneficial for early treatment before the disease develops or progresses too far and will result in greater longevity (Katsanis & Katsanis, 2013). Testing for ARC may lead to the diagnosis of a highly likely predisposition to AD because of its strong link to the ApoE4 allele. With the ApoE4 gene the mean age to develop AD is 68 with a 91% chance for homozygotes, 76 years old with a 47% chance for heterozygotes, and 84 years old with 20% for people who do not carry the allele (Zhong, et al., 2016). There is an ethical dilemma when it is revealed that a patient has the ApoE4 allele, since exposing genetic risk is a complex issue, as it not only shows risk for the patient but also to the patient’s family member who may also have the allele. They would have to reveal to their relatives that they have the ApoE4 allele and that they should also get tested. The cost of testing for the just one allele would be low since it would not require a large amount of DNA sequencing, a simple PCR test would be sufficient. It also reveals to the patient that they may pass on this gene to their offspring, which might become a burden on them from having any future children. If they are not in a relationship they would also feel pressure that they have to reveal that they are carriers to future partners (Arribas-Ayllon, 2011).

There is no clear benefit to early disclosure of the predisposition of getting AD to young adults because there is no medical intervention available. The psychological harm from the revelation of being a carrier of the ApoE4 allele outweigh the benefits of disclosure. Clinicians may feel that they don’t need to need to reveal to a patient their risk of AD when they have a genotypic test for ARC. As of now, there are no guidelines for clinicians on deciding whether the association between a gene and disease have “sufficient clinical validity and usefulness to justify disclosure (Green, et al., 2009).”

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