This work describes the identification and characterization of a novel APP mutation E682K from a single case of early onset AD. The index patient is a Belgian female; she was diagnosed as probable AD at the age of 49 years. Neuropsychological evaluation, CSF biomarkers (Aβ42
, total-tau and phospho-tau), amyloid imaging (using 11
C-PIB PET) as well as magnetic resonance imaging of brain all confirmed the diagnosis of AD. The patient's father developed late onset AD, while the brother is an asymptomatic mutation carrier currently at the age of 53, and there is no extended familial information. This raised the interesting question whether the identified mutation is a benign polymorphism or should be considered a genuine genetic risk factor for disease. The issue of rare genetic variants contributing moderate risk to disease is hotly debated in genetic research, as it is very difficult to detect such mutations while they are likely to contribute significantly to the total genetic risk for disease (Singleton et al, 2010
). We hypothesized that the APP E682K mutation could have variable penetrance, which has previously also been proposed for disease causing mutations in PSEN2
, carriers of which display a strong variation in age of onset (Sherrington et al, 1996
) and decided to use functional assays to test whether the identified mutation could be considered as pathogenic or not.
Our analysis showed that this novel mutation increased full length Aβ release and also the Aβ1–42
ratio to similar extents as found with a previously well characterized disease associated APP A692G or ‘Flemish’ mutation. While the observed effects on Aβ processing suggest the possibility that this novel mutation is pathogenic, the genetic support is inconclusive. According to the criteria discussed by Guerreiro and coworkers (Guerreiro et al, 2010
), the APP E682K mutation was found in a single case of early onset AD. This mutation was absent from 940 healthy controls and affects Aβ generation. Although strictly spoken a second case with this mutation needs to be identified as independent confirmation (Guerreiro et al, 2010
), we propose that the accumulated evidence is clearly in favour to classify this mutation as probable pathogenic
. Further follow up of the brother of the index case could yield final confirmation, but because of the study design, we are not allowed to contact the brother for further investigation. Therefore, we will have to wait for additional cases from other investigations in the future to classify this mutation as definitively pathogenic. Nevertheless, the functional analysis of this mutation provided an important and interesting novel insight in the biological significance of the β′-site processing of APP, which appeared quantitatively much more important for normal APP metabolism than previously thought.
Our data indicate indeed that processing at this site should be considered anti-amyloidogenic, counteracting partially the amyloidogenic β-secretase cleavage of APP. In agreement with this assumption, rodents, which tend to cleave their endogenous APP at the β′-site (De Strooper et al, 1995
), do not spontaneously develop amyloid plaques. The functional significance of β′-site cleavage of APP by BACE1 has indeed been debated since a long time. On the one hand, it was previously suggested that this cleavage is a minor part of physiological APP processing (Creemers et al, 2001
). Our new study now clarifies that this is not the case and that about one-fourth of the major APP CTF species is generated by this pathway under our experimental conditions. Second, it was reported that N-terminal truncation of Aβ may enhance the aggregation properties as well as the cytotoxic effects of Aβ (Demeester et al, 2001
) and that in sporadic AD cases, significant amounts of Aβ11–40/42
species were found in the plaques in the brain (Huse et al, 2002
; Liu et al, 2006
). Therefore, β′-site cleavage was proposed to contribute to AD pathogenesis (Huse et al, 2002
; Liu et al, 2006
). However, our data argues for a protective role of β′-site cleavage, as blocking this cleavage disrupted the balance of APP processing by BACE1 leading to increased full length Aβ. The fact that the β′-site mutation E682K is associated with early onset AD speaks clearly in favour of this argument.
Of note, our data suggest additional effects of the E682K mutation on γ-secretase activity as well. APP carrying the E682K mutation increased significantly the Aβ1–42
ratio in cell-based assays. This effect occurs at the γ-secretase level because expressing a C99 construct that bypasses the ectodomain shedding step needed for γ-secretase (Lichtenthaler et al, 1999
) led to a similar increase in this ratio. It has been recently reported that a substrate inhibitory domain (ASID) located at the middle of Aβ (17–23) is modulating γ-secretase activity by binding to an allosteric site within the γ-secretase complex and that the ‘Flemish’ mutant C99 increased γ-secretase activity via disruption of this ASID (Tian et al, 2010
). The E682K mutation is located at the Aβ11
site, which is not part of the previously delineated ASID, but had nevertheless a significant effect on Aβ1–40
levels and little effects on Aβ1–42
levels. We suggest the possibility that the interaction domain between γ-secretase and C99 is more extended than ASID and mutations such as the E682K mutation, as well as other similar substitutions, may be considered as new tools to investigate the possible allosteric modulation of γ-secretase by APP substrate.
The current data indicate that BACE1 cleaves APP at two distinct sites and that both are physiologically significant events in APP processing. So far, BACE1 is the most attractive drug target for AD treatments, and the evaluation of inhibitors under development for treatment of the disease focuses on the effect of blocking the β- site cleavage. This could be problematic because BACE1 inhibitors will also affect β′-site cleavage. Kinetic data showed that the BACE1 has a much higher enzymatic efficiency towards peptide containing the β-site than peptide containing the β′-site sequence (Yang et al, 2004
), which raises a theoretical possibility that BACE1 inhibitors may have differential inhibition effects on the two distinct sites depending on the concentrations used. This could cause relative shifts from β′- to β-site cleavage, as observed with the current mutation. We thus propose that inhibitor drugs should be carefully monitored for their dose-effect on both BACE1 cleavage sites and to measure, therefore, the effects on Aβ11–40/42
of any such drug when used in patients.
Finally it is likely that in the near future, increasing exon- and also exome-based sequencing efforts will increase the number of putative pathogenic mutations identified without further conclusive segregation-based evidence in a single family. Functional analysis of such mutations as demonstrated here will allow to determine the putative pathogenic nature of some of these mutations as more extensively discussed in (Guerreiro et al, 2010
). Such functional study may also be crucial to fill part of the existing gap in genetic research with regard to the postulated low frequency, moderate risk genetic loci that are now very difficult to identify and were called the ‘dark matter of disease risk’ (Manolio et al, 2009
; Singleton et al, 2010