How proteomic ApoE serotyping could impact Alzheimer’s disease risk assessment: genetic testing by proteomics

Abstract

Humans have three major apolipoprotein E (ApoE) alleles (APOE; ε2, ε3 and ε4) that produce three ApoE protein isoforms. The ε2 allele encodes the ApoE2 isoform (Cys112, Cys158), whereas ε3 encodes the wild-type ApoE3 isoform (Cys112, Arg158) and ε4 encodes the ApoE4 isoform (Arg112, Arg158). Because the type of ApoE expressed is related to sporadic Alzheimer’s disease risk and familial hyperlipidemia, many clinical studies have utilized ApoE typing in recent years. ApoE serotyping is based on the correlation between ApoE genotype and isoform; it is therefore possible to determine the genotype from the blood ApoE isoform combination. Serotyping ApoE using mass spectrometry promises highly accurate results while requiring minimal amounts of blood and reagents, resulting in lower costs, which suggest that proteomic-based ApoE serotyping may eventually become a routine clinical laboratory test. Not limited to ApoE, proteomic analysis of human samples could be used to intentionally determine – and perhaps unintentionally reveal – personal genetic information.

Keywords::

• ApoE2 isoform
• ApoE4 isoform
• APOEε2 allele
• APOEε4 allele
• apolipoprotein E
• dementia risk
• familial hyperlipidemia
• genetic testing
• proteomics
• sporadic Alzheimer disease

What is apolipoprotein E serotyping?

Usually, cell surface antigens of microorganisms, viruses or cells derived from individuals are subjected to serotyping (a typical example is ABO blood group typing). In some cases, secreted proteins, such as botulinum toxins, are subjected to serotyping. Typing of serum apolipoprotein E (ApoE) isoforms is termed ‘ApoE serotyping’. Recent technical advances in mass spectrometry (MS) have made possible the detection of single amino acid substitutions, thus enabling the typing of a wide variety of proteins. For example, surface-enhanced laser desorption/ionization–time-of-flight MS was utilized to evaluate the variants of serum transthyretin to diagnose familial amyloid polyneuropathy [1].

ApoE typing & its role in the diagnosis of Alzheimer’s disease & other conditions

At present, ApoE types are usually determined using genetic analysis. A 1995 consensus report on the role(s) of APOE genotyping in the diagnosis and the prediction of the future risk of Alzheimer’s disease (AD) concluded that APOEε4 is strongly associated with AD and is an important risk factor for the development of the disease [2,3]. However, the report recommended against the use of APOE genotyping for routine clinical diagnosis. The report did, however, acknowledge that other uses for APOE genotyping in the management of AD might be identified in coming years. Similar recommendations were published in 2007 as part of the third Canadian Consensus Conference on the Diagnosis and Treatment of Dementia [4]. However, the report considered APOE genotyping as potentially useful for stratifying patients with mild cognitive impairment at risk for progression [4] and for evaluating therapeutic efficacy in the context of clinical research [4].

On the basis of these recommendations, tertiary care centers now routinely determine APOE genotype as part of the medical evaluation of elderly people with cognitive dysfunction/dementia as well as neurologically healthy individuals in the context of clinical and epidemiological research [3]. For example, APOE genotyping of participants in an epidemiological study by Crane et al. [5] was performed as a matter of course.

Because the type of ApoE expressed is believed to be an indicator of AD risk as well as having pathological roles in several clinical conditions [6], many clinical studies have utilized ApoE typing in recent years. APOEε2 (ApoE2) is a causative gene of type III familial hyperlipoproteinemia, despite its low penetrance. Moreover, in addition to the major ApoE2/E3/E4 isoforms, many other pathogenic ApoE isoforms, such as ApoE-Sendai (Arg145Pro) of lipoprotein glomerulopathy, have been registered to date with Online Mendelian Inheritance in Man [7].

Which ApoE isoforms should be serotyped?

Although a distinct relationship between AD risk and the APOEε4 allele has been established, serotyping should not be limited to determining the presence or the absence of APOEε4 (ApoE4) alone. The risk of AD differs between APOEε4 homozygotes and heterozygotes [8]; thus, distinguishing APOEε4 homozygous and heterozygous carriers is important. In the case of APOEε4 heterozygotes, the other ApoE types an individual carries becomes important because the APOEε2 (ApoE2) allele protects against late-onset AD (even if the other allele is APOEε4 [8]), although APOEε2 itself is linked to familial hyperlipoproteinemia. With respect to APOE allele prevalence (in the United States, the allele frequency of APOEε2 is approximately 0.07, while APOEε4 allele frequency is approximately 0.15 and the most common allele is APOEε3 [9]), ApoE serotyping methods must be capable of accurately typing the ApoE2/E3/E4 isoforms, as well as their homozygous/heterozygous combinations. Other rare ApoE mutations are known to cause familial hyperlipoproteinemia (for example, ApoE-Christchurch (Arg136Ser)). Thus, from an epidemiological standpoint, ApoE serotyping should ideally be capable of detecting rare ApoE isoforms. In addition to AD and hyperlipoproteinemia, serotyping methods capable of such performance would be useful in diagnosing other diseases involving APOE mutations, such as familial lipoprotein glomerulopathy, dysbetalipoproteinemia, hypercholesterolemia and hypertriglyceridemia (as registered in Online Mendelian Inheritance in Man).

Conventional methods & MS in ApoE analysis & typing

Historically, scientists have analyzed ApoE and its isoforms by electrophoresis. For example, triglyceride-rich lipoproteins prepared from blood samples were typically analyzed by isoelectric focusing (and later by 2D electrophoresis). An ApoE4-specific antibody has been developed [10] and used in a commercially available ELISA kit. However, this ELISA only permits determination of the presence or absence of ApoE4 and not the other ApoE types.

Due to sequence variations, the digestion of most ApoE isoforms with trypsin results in peptide fragments that differ substantially in mass from those of wild-type ApoE3 because trypsin specifically cleaves the peptide bond between the carboxyl group of Arg or Lys residues and the amino group of the adjacent residue. ApoE isoforms containing mutations involving Arg or Lys residues (such as ApoE2 and ApoE4) will produce fragments of differing mass compared with isoforms involving the substitution of a single amino acid other than Arg or Lys. The substantial mass differences resulting from trypsin digestion in such cases is termed tryptic fragment length polymorphism (TFLP), which is similar in concept to restriction fragment length polymorphism (RFLP) in molecular biology. Studies focusing attention on digestion-related mass differences in proteins were reported as early as 1982 [11]; accurate mass analysis of peptides was difficult at that time; however, the detection of a novel ApoE mutant isoform based on tryptic peptide analysis was reported in 1987 [12]. More recent studies have used MS-based proteomic analyses to type and quantitatively determine ApoE isoforms. MS allows accurate mass determinations of ApoE tryptic peptides and, therefore, facilitates accurate identification of ApoE isoforms and homozygous/heterozygous status. One recent report described the use of stable isotope labeling tandem MS for isoform-specific relative quantitation of ApoE4 [13]. This approach is based on the comparative measurements of cells labeled in culture with amino acids incorporating stable isotopes. Another study attempted MS-based quantification of ApoE isoforms by labeling full-length ApoE with a stable isotope [14]; however, this method still requires prior APOE genotype information. Nishimura et al. [15] reported a novel ApoE serotyping method involving protein resequencing that requires no stable isotope labeling or genome analysis. In theory, this method should be capable of identifying not only ApoE2/E3/E4 isoforms and their homozygous/heterozygous combinations but also mutations that do not involve Lys or Arg residues.

Impact of proteomic ApoE serotyping on AD risk assessment

Typing by MS promises highly accurate results while requiring minimal amounts of blood and reagents, resulting in lower costs. The relatively high cost of MS instruments, however, makes MS-based ApoE serotyping most suited for use as a large, laboratory-centered clinical test, such as those performed during medical checkups. Because serum is a universal starting material for clinical testing, ApoE serotyping of serum (and perhaps plasma also) by MS could be easily integrated into existing clinical laboratory test programs. Indeed, Simon et al. [16] reported the quantitation of total ApoE and the ApoE4 isoform in plasma in a cohort of 669 individuals. Nishimura et al. [15] reported that one-tenth of a 5 µl serum sample is sufficient for ApoE serotyping by liquid chromatography–tandem MS. These data suggest that proteomic-based ApoE serotyping may eventually become a routine clinical laboratory test more widely utilized than DNA genotyping. Along this line, it is noteworthy that Panza et al. [17] suggested the role of different common APOE polymorphisms in controlling serum APOE level, which in turn has a potential value as a risk factor for AD [18].

In the case of hyperlipidemia, a kind of light-hearted introduction may be useful. Enabling type III hyperlipoproteinemia patients to have their ApoE2-carrier status determined at the same time as their cholesterol and triglyceride levels are measured would be highly advantageous because it would inform them of their need for intensive cholesterol-lowering therapy to reduce the future risk of fatal cardiovascular diseases. However, is ApoE serotyping always useful in diagnosing the risk of AD? Effective prevention drugs remain unavailable for those at risk for AD, which has resulted in controversy regarding the role of routine ApoE typing in AD diagnosis. Nevertheless, the relationship between the risk of AD and ApoE4 has been clearly established. ApoE4 typing is known to have other health benefits as well. For example, young persons who are APOEε4 positive should be advised to avoid participation in contact sports or other activities that expose them to the risk of traumatic brain injury [19,20].

In conclusion, because ApoE is a pleiotropic molecule, proteomic ApoE serotyping could be considered a pleiotropic testing method. It seems likely that proteomic techniques will very soon enable cheaper and easier ApoE serotyping than previous methods. The potential for MS is, of course, not limited to ApoE typing. In the very near future, proteomic analysis of human samples by MS will determine personal genetic information and enable its dissemination far more widely than is now possible, which in turn may lead to a significant number of incidental findings. It is time for clinical geneticists and scientists in the proteomics field to begin serious discussions of this issue.

Financial & competing interests disclosure

This study was supported by a Grant-in-Aid for Young Scientists (B) (No. 24790548 (13059483)) from JSPS KAKENHI. The funder had no role in the study design, data collection, analysis, decision to publish, or preparation of the manuscript. The authors acknowledge writing assistance by Will Costain of Forte Science Communications (Japan). The authors have no other relevant affiliations or financial involvement with any organization or entity with a financial interest in or financial conflict with the subject matter or materials discussed in the manuscript. This includes employment, consultancies, honoraria, stock ownership or options, expert testimony, grants or patents received or pending, or royalties.