Apolipoprotein E is a class of proteins that play a major role in the metabolism of dietary fat. This protein is considered to be linked to Alzheimer’s disease and cardiovascular disease. It is an important regulator of blood levels of both cholesterol and triglycerides. It plays an integral part in the transport of these fats in the body (Sigurdsson, 2016). Apolipoprotein is polymorphic with three major alleles E2, E3, and E4; and five hypothetical ones. These alleles can differ by only one or two amino acids at positions 112 and 158 (Sigurdsson, 2016).
These alter the structure and function, which can cause potential consequences.
E3 is the common allele in the general population that does not influence risk. E4 allele is linked to Alzheimer’s disease, Dementia, and atheroscierosis. One copy of this allele increases risk by 2 to 3 times. Two copies of this allele increase the risk of up to 12 times (Dacks, 2016). E2 allele is linked to hyperlipoproteinemia type III and a rare type, this includes one copy for on-set Alzheimer’s.
Everyone has two copies of this gene and it’s the combination that will determine your ApoE genotype (Dacks, 2016). For this case study, Dr. Carl Yamashiro explained the hypothetical objective, “The interactions between the hypothetical and known alleles will have the hypothetical ones mimic E3 in terms of phenotypes known to be associated with the known alleles.” Knowing the situation the types of technologies that are cable of genotyping all eight of ApoE alleles would be: RT-PCR using TaqMan probe or a high-resolution melt, DNA Microarrays, and Next-generation sequencing using Roche 454, Illumina or SOLiD.
Real-Time Polymerase Chain Reaction is a laboratory technique that is based on the standard polymerase chain reaction. Standard PCR is the amplified DNA or amplicon that is detected in an endpoint analysis, and in real-time PCR is the accumulation of amplification product measured as the reaction progresses.
It will quantify during each cycle (Bio-Rad Laboratories, 2019). In Zhong et al. article, the genotyping method used along with one TaqMan probe for the polymorphic alleles, which are recognized by site-specific PCR primers. It was obtained by, one-doubled-dye oligonucleotide TaqMan probe. This probe was a FAM fluorophore that attached to its 5’ end and a BHQ quencher molecule attached to its 3’ end. It will then hybridize to the template strand by its complementary sequences. During the PCR amplification step, the Taq polymerase will degrade during strand extension, resulting in the separation of the fluorophore and the quencher will allow the excitation of the fluorophore from the laser (Zong, et al., 2016).
Some advantages when using Real-Time PCR are that it’s automated and less labor-intensive than standard PCR. It is also rapid, sensitive, specific, robust, cost-effective, high-throughput application, and efficient. These are advantages that are needed in order to be able to genotype all 8 alleles. A disadvantage would be using an RT-PCR instrument versus Standard PCR using gel electrophoresis. The instrument and reagents can be more costly than the standard.
Real-Time Polymerase Chain Reaction using heat resolution melting can also be used to genotype all eight ApoE alleles. This technique is used for the detection of
mutations, polymorphisms and epigenetic differences in the double-stranded DNA sample (Wikipedia). Its method uses fluorescent dyes that will bind specifically to double-stranded DNA and will fluoresce brightly once bonded. Once the sample is heated up, the two strands of DNA will melt apart. The instrument includes a camera, which will measure the fluorescence and plot the data on a graph. The melt curve graph will show the level of fluorescence versus the temperature.
This is significant when comparing DNA that has a mutation in the region being amplified because the tiny difference is monitored in high resolution and can be accurately documented (Wikipedia). I think this technology would be able to genotype all of the alleles and possibly detect the alleles that have complexities to them. Some advantages for this technology are that it is a simple, rapid, inexpensive, accurate and sensitive method for genotyping (Zhan, et al., 2014).
It has a high-throughput with a closed-tube approach for screening. This will reduce the risk for contamination (Zhan, et al., 2014). A disadvantage for this technology is that the sensitivity and specificity in an individual clinical diagnostic setting are variable (Li, et al., 2011).
The second technology which could genotype all eight of the ApoE alleles would be DNA Microarrays. This technology will be able to determine whether the DNA from the individual will contain a mutation in their genes. Each chip includes thousands of short synthetic, single-stranded DNA sequences, which together add up to the normal gene in question. There are two major microarrays used, but the oligonucleotide microarray would specifically work in this case study. The oligonucleotide microarray, in particular, could genotype all of the alleles, and provide a higher target specificity.
Oligonucleotide microarrays are short DNA oligonucleotides that are spotted on the array. These probes will map the different regions of the gene, and target specific genes for better discrimination of highly similar targets such as splice variants or gene families. A common product used would be Affymetrix. It uses photolithography to manufacture short single strands of DNA onto a 5-inch square wafer. Some advantages of using this type of technology include speed, specificity, and reproducibility.
Specificity and reproducibility are important due to the way the oligonucleotide sequences will be printed onto the chip. The sequence will be designed to be complementary to a target gene except for a single base mismatch. This mismatch strategy along with the multiple sequences for each gene increases specificity and minimizes the effects of non-hybridization and background signal.
Some disadvantages that can occur when using this type of microarrays are affordability and flexibility. This type of array can have expensive equipment to carry out each process. And the Gene chips themselves can be expensive. As for flexibility, the processes can overlap and needs to be timed out for an appropriate workflow.
Finally using Next Generation Sequencing (NGS) would the last optimal choice for being a technology that can perform the task of genotyping all the ApoE alleles. There are three main platforms for this type of technology, which are Roche 454, Illumina, and Life Technologies/SOLiD. NGS was developed to moderate cost, be user-friendly and manage large amounts of data. They are used for extensive gene sequencing, for disease mapping, for quantifying RNA sequencing, and population genetic studies ( ). NGS delivers a higher throughput at a faster turnaround time. In NGS methods, “A whole genome, or targeted regions of the genome is randomly digested into small fragments. These will get sequenced and are then either aligned to a reference genome or assembled”( ). The aligned the fragments of one or more individuals will be a reference genome, and SNP calling will identify the variable sites. So the genotype calling will determine the genotype for each individual at each site.
In the Roche 454 platform, the detection technique it is based on is emulsion PCR and pyrophosphate detection techniques ( ). The “sheared DNA fragments are ligated to specific oligonucleotide adapters, which subsequent to the binding of each DNA fragment to a fragment-carrying bead”( ). These beads are captured and separated by dilution then later deposited into a picotiter-plate well. The sequence reaction method used here is pyrosequencing in high-density picoliter reactors.
This method is different from other platforms. Some advantages with this platform are the system has longer reads. It can generate over 1 million individual sequence reads with reading lengths over 400 bases during a 10-h timespan. Another advantage would be the library construction is automated and that reduces manpower. But one of the disadvantages is the high cost of reagents, which averages out to be $12.56 X 10-6 base pair. It also has a relatively high error rate in terms of poly-based longer than 6 base pair ( ).
As for the Illumina, it is the first short-read sequencing platform. It uses “an array technique to achieve cloning-free DNA amplification. It sequences by synthesis with reversible terminator chemistry. “For sequencing, the reaction mixtures contain primers, DNA polymerase and four reversible terminator nucleotides, which are labeled with different fluorescent dye, are supplied to the flow cell”.
The flow cell is determined to the fluorescent emission, and then the camera records the signal. In the next step, the reversible terminator is unblocked and the fluorescent dye label is removed from the base. This is so that the new nucleotide can be incorporated and the new base can be detected. This takes longer than the Roche 454, by be completed within 2.5 days to generate 50 million reads per flow, with a read length of 36 bases. The advantages of this technology allow for shorter reads, more data to be generated, low error rate, and it is cost effective. One disadvantage would be the longer turnaround time for the process to be completed.
Finally the SOLiD, the technique is similar to the Roche 454 but its sequencing reaction is done by ligation chemistry. It is similar in the sense that the DNA is sheared into fragments then ligated to oligonucleotide adapters, which attach beads and clonally amplified by emulsion PCR (). Originally the SOLiD was able to output 35 bp reads, which led to high accuracy. With further upgrades and improvements, the “SOLiD 5500xL has improved read lengths, accuracy, and data output of 85 bp. A complete run would be finished in 7 days, and the cost is about $40 X 10-9 per base of reagent use”. The disadvantage would be the shorter read lengths and resequencing.
I would choose the Real-Time PCR using TaqMan capabilities for this study out of the three, reasons being for the advantages mentioned. This type of platform is rapid, cost-effective, specific, and sensitive. The others have all the similar advantages, but RT-PCR would cost a lot less to test compared to the other platforms. This platform is ideal because it can visualize the primers for each allele for ApoE in real-time. As for Microarrays and NGS platforms take more time to set up and run through their processes compared to RT-PCR. They also require multiple instruments for different processes, which can be more costly.
A plan to develop a test using this technology would mean developing a TaqMan that can detect all 8 alleles, and also is applicable to PCR. As we know TaqMan requires the use of a specialized probe with two-fluorophores labels, which hydrolyzed during the reaction. I would think specialized probes and primers to detect the 8 alleles would need to be developed. According to Kamau, to develop this an SNP sequence to Applied Biosystems (AB) needs to be submitted for analysis of their suitability to be used in an SNP assay. After submitting the sequences to a custom TaqMan assay design tool, which can be accessed on the AB website.
The result will be the number of sequences returned for the SNP assay by identifying which were potentially good candidates. Here we hope that all alleles can be identified. The next step would be developing the concentration for the TaqMan genotyping assay mix. The mix would contain a forward and reverse primer. Also taking into account the probes, they must contain a non-fluorescent “quencher and a minor groove binder moiety which allows for shorter probe sequences to be designed”(Kamau, 2012).
Once the master mix is designed, the DNA template must be designed by software that analyzes the before and after fluorescence levels. The calculated normalized dye fluorescence will act as a function of the cycle number needed for the alleles. The primers are designed by “Primer Express 3.0 software”(Kamau, 2012).
This will amplify amplicons of interest. In order to do this, an amplified reaction mixture must be made by Applied Biosystems, which usually is a buffer of “ 1XPCR gold buffer with MgCl2” (Kamau, 2012). Lastly, the cycling conditions will need to be determined by the cycling threshold. The amplification of PCR fragments will also need to be confirmed.
The potential ethical issues regarding the ApoE4 allele were to be associated with an increased risk of a late-onset form of Alzheimer’s disease. The ethical issue looked at was tests showed susceptibility, which was different from predictive testing. Physicians were cautious against wanting to do susceptibility testing because there are no treatment options for the disease. This could have the potential for causing more health issues. In this article…., susceptibility and predictive testing were compared ethically the same.
In 1997, a consensus meeting was held about ApoE testing where five conferences claimed this allele was not appropriate because sensitivity and specificity were too low. But the argument remained; ApoE genotyping was “ripe for misunderstanding among families and increased social cost and psychological burdens”. The cost of genotyping and sequencing could reduce since the number of genetic tests for susceptibility is likely to increase because of the “pressure on the state health-care systems providing testing in the future”.
Also further testing on the behavioral and environmental standpoint. People/Clinicians would want testing done to see if their behaviors or if their environment they are in as any effect. According to Howe, he discussed how if people were to do the testing and tested for the allele that they can see a professional like a psychiatrist who could sit down with their patient and explain what the result means. And further, if they had stresses could come frequently to discuss it more. So, in the long run, it could protect people from depression or other illnesses. When taking account the benefit of early detection of cancer versus informing a younger individual that they have a genotype indication a possibility of contracting early-onset Alzheimer’s is what I believe the personal choice to do what they want with the information.
Detecting early cancer is saying that the person has it or going to get it, and there is a need to do something to preserve their life. So when there is a possibility of contracting early-onset Alzheimer’s disease, if a person has a small chance of having the gene then that person could take preventative measures.