Overview

Pathogenicity: Cerebral Amyloid Angiopathy : Pathogenic
ACMG/AMP Pathogenicity Criteria: PS3, PM1, PM2, PM5, PP1, PP2, PP3, PP4
Clinical Phenotype: Cerebral Amyloid Angiopathy
Reference Assembly: GRCh37/hg19
Position: Chr21:27264168 G>A
dbSNP ID: rs63750579
Coding/Non-Coding: Coding
DNA Change: Substitution
Expected RNA Consequence: Substitution
Expected Protein Consequence: Missense
Codon Change: GAA to AAA
Reference Isoform: APP Isoform APP770 (770 aa)
Genomic Region: Exon 17

Findings

This mutation was originally identified in four Italian families from Southern Lombardy who had a form of familial cerebral amyloid angiopathy (CAA). Affected individuals presented with recurrent headaches and multiple strokes followed frequently by epilepsy and cognitive decline (Tagliavini et al., 1999). The mutation was shown to cosegregate with disease in at least two of the families (Bugiani et al., 2010).

The variant was also identified in a French woman and her brother who both suffered from hematomas (Sellal et al., 2017).  The proband had visual impairment, developed migraines at a young age, and had progressive memory loss starting at age 60. Both carriers were homozygous for APOE3. Their mother, who was not genotyped, had a stroke and died at age 63.

Based on the 16 carriers with available information, mean age at clinical onset was 54 years (Sellal et al., 2017).

This mutation was absent from the gnomAD variant database (v2.1.1, Oct 2021).

Neuropathology

Neuropathological data for one Italian carrier revealed Aβ immunoreactivity in the walls of leptomeningeal and parenchymal vessels and in the neuropil (Bugiani et al., 2010). No phosphorylated tau, neurofibrillary changes, or neuritic plaques were observed.

MRI brain imaging of several Italian patients showed small to large hematomas, subarachnoid bleeding (and/or calcifications which appear similar to subarachnoid hemorrhages, see Sellal et al., 2017), scars with hemosiderin deposits, small infarcts, and leukoaraiosis. Aβ immunoreactivity was detected in the walls of leptomeningeal and parenchymal vessels and in the neuropil. This disease is distinguished from Alzheimer's disease with CAA by the absence of neurofibrillary changes and neuritic plaques (Tagliavini et al., 1999; Bugiani et al., 2010).

Interestingly, in both French patients, serpentine cortical calcifications were identified by MRI and CT scans, a feature observed in carriers of the APP D694N (Iowa) mutation, but not other APP mutations (Sellal et al., 2017).  In addition, the siblings had occipital hematomas and microbleeds. In the proband, sequelae of cerebellar infarction were evident. Moreover, biomarkers in her cerebrospinal fluid were consistent with AD, including decreased levels of Aβ42 and increased levels of tau and phospho-tau.

Biological Effect

As assessed in cultured PC12 cells, E693K peptides Aβ42, and particularly Aβ40, are toxic (Murakami et al., 2003). The mutant peptides, a.k.a. E22K, aggregate faster, displaying a shortened, sometimes undetectable, lag phase (Hatami et al., 2015, Yang et al., 2018, Illes-Toth et al., 2021). A study using global kinetic analysis suggested this acceleration, at least for Aβ42 peptides, stems primarily from enhanced secondary nucleation on the surface of existing fibrils (Yang et al., 2018).

E22K Aβ42 forms longer fibrils and more loosely packed aggregates than wildtype Aβ (Yang et al., 2018), and similarly, E22K Aβ40 fibrils are less bundled together and form more amorphous aggregates than wildtype Aβ40 (Hatami et al., 2015). The self-assembly of mutant Aβ42 has been studied in detail (e.g., Illes-Toth et al., 2021), with some studies suggesting it yields a parallel β-sheet structure (e.g., Masuda et al., 2008), and others indicating an antiparallel β-sheet with less α-helicity than wildtype Aβ, possibly due to destabilization of the hydrogen bonding network required for maintaining α-helical structure (Hubin et al., 2015, Davidson et al., 2022).

An increase in  solvent exposure at the C-terminus has been predicted to fuel fibril formation (Davidson et al., 2022). Although E22K has two fewer negative charges than wildtype Aβ, at least one study suggests that the large size of the lysine side-chain, rather than its charge, may be key to its effect on aggregation kinetics (Yang et al., 2018).

Mutant Aβ40 fibrils have been reported as being thermostable (Lee et al., 2022) and mutant Aβ42 oligomers appear to be more internally stable than wildtype oligomers (Kassler et al., 2010). Nevertheless, the β-sheet structure of mutant Aβ42 fibrils appears to have structural plasticity, changing in a pH-dependent manner (Hubin et al., 2015).

A few studies have examined how E22K may cause cytotoxicity via its effects on cell membranes. One in vitro study, for example, suggested E22K oligomers destabilize membranes and disrupt their integrity (McKnelly et al., 2022). Moreover, a structural modeling study predicted that the mutant Aβ peptide partitions into membranes more than wild type Aβ peptides (Kim and Bezprozvanny, 2023). This is expected to disrupt the normal function of these peptides in the endosomal lumen where they may play important roles in synaptic and neuronal function. Also of note, E693 lies within a cholesterol-binding site as determined by NMR resonance spectroscopy and site-directed mutagenesis (Barrett et al., 2012).

E693K also appears to have an effect on the production and degradation of Aβ peptides. HEK cells transfected with mutant APP produced significantly lower amounts of Aβ42, while levels of Aβ40 were similar to those produced by wild-type APP. Therefore, the ratio of Aβ42/Aβ40 was reduced (Nilsberth et al., 2001). Moreover, E22Q aggregates appear to be resistant to proteolytic degradation by neprilysin, a peptidase that degrades Aβ in the brain (Tsubuki et al., 2003).

E693K's PHRED-scaled CADD score, which integrates diverse information in silico, was above 20, suggesting a deleterious effect (CADD v.1.6, Oct 2021).

Pathogenicity

Cerebral Amyloid Angiopathy : Pathogenic*

*Although not AD, the condition associated with this variant is inherited in an autosomal dominant manner, so its pathogenicity was classified using the ACMG-AMP guidelines.

This variant fulfilled the following criteria based on the ACMG/AMP guidelines. See a full list of the criteria in the Methods page.

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References

Mutations Citations

1. APP D694N (Iowa)

Paper Citations

1. Tagliavini F, Rossi G, Padovani A, Magoni M, Andora G, Sgarzi M, Bizzi A, Savoiardo M, Carella F, Morbin M, Giaccone G, Bugiani O. A new βPP mutation related to hereditary cerebral haemorrhage. Alzheimer's Reports. 1999; 2(S1):S28.
2. Bugiani O, Giaccone G, Rossi G, Mangieri M, Capobianco R, Morbin M, Mazzoleni G, Cupidi C, Marcon G, Giovagnoli A, Bizzi A, Di Fede G, Puoti G, Carella F, Salmaggi A, Romorini A, Patruno GM, Magoni M, Padovani A, Tagliavini F. Hereditary cerebral hemorrhage with amyloidosis associated with the E693K mutation of APP. Arch Neurol. 2010 Aug;67(8):987-95. PubMed.
3. Sellal F, Wallon D, Martinez-Almoyna L, Marelli C, Dhar A, Oesterlé H, Rovelet-Lecrux A, Rousseau S, Kourkoulis CE, Rosand J, DiPucchio ZY, Frosch M, Gombert C, Audoin B, Miné M, Riant F, Frebourg T, Hannequin D, Campion D, Greenberg SM, Tournier-Lasserve E, Nicolas G. APP Mutations in Cerebral Amyloid Angiopathy with or without Cortical Calcifications: Report of Three Families and a Literature Review. J Alzheimers Dis. 2017;56(1):37-46. PubMed.
4. Murakami K, Irie K, Morimoto A, Ohigashi H, Shindo M, Nagao M, Shimizu T, Shirasawa T. Neurotoxicity and physicochemical properties of Abeta mutant peptides from cerebral amyloid angiopathy: implication for the pathogenesis of cerebral amyloid angiopathy and Alzheimer's disease. J Biol Chem. 2003 Nov 14;278(46):46179-87. Epub 2003 Aug 27 PubMed.
5. Hubin E, Deroo S, Schierle GK, Kaminski C, Serpell L, Subramaniam V, van Nuland N, Broersen K, Raussens V, Sarroukh R. Two distinct β-sheet structures in Italian-mutant amyloid-beta fibrils: a potential link to different clinical phenotypes. Cell Mol Life Sci. 2015 Dec;72(24):4899-913. Epub 2015 Jul 21 PubMed.
6. Yang X, Meisl G, Frohm B, Thulin E, Knowles TP, Linse S. On the role of sidechain size and charge in the aggregation of Aβ42 with familial mutations. Proc Natl Acad Sci U S A. 2018 Jun 26;115(26):E5849-E5858. Epub 2018 Jun 12 PubMed.
7. Illes-Toth E, Meisl G, Rempel DL, Knowles TP, Gross ML. Pulsed Hydrogen-Deuterium Exchange Reveals Altered Structures and Mechanisms in the Aggregation of Familial Alzheimer's Disease Mutants. ACS Chem Neurosci. 2021 Jun 2;12(11):1972-1982. Epub 2021 May 14 PubMed.
8. Masuda Y, Nakanishi A, Ohashi R, Takegoshi K, Shimizu T, Shirasawa T, Irie K. Verification of the intermolecular parallel beta-sheet in E22K-Abeta42 aggregates by solid-state NMR using rotational resonance: implications for the supramolecular arrangement of the toxic conformer of Abeta42. Biosci Biotechnol Biochem. 2008 Aug;72(8):2170-5. Epub 2008 Aug 7 PubMed.
9. Davidson DS, Kraus JA, Montgomery JM, Lemkul JA. Effects of Familial Alzheimer's Disease Mutations on the Folding Free Energy and Dipole-Dipole Interactions of the Amyloid β-Peptide. J Phys Chem B. 2022 Oct 6;126(39):7552-7566. Epub 2022 Sep 23 PubMed.
10. Lee MC, Liao YH, Chen SH, Chen YR. Amyloid-β40 E22K fibril in familial Alzheimer's disease is more thermostable and susceptible to seeding. IUBMB Life. 2022 Aug;74(8):739-747. Epub 2021 Nov 1 PubMed.
11. Kassler K, Horn AH, Sticht H. Effect of pathogenic mutations on the structure and dynamics of Alzheimer's A beta 42-amyloid oligomers. J Mol Model. 2010 May;16(5):1011-20. Epub 2009 Nov 12 PubMed.
12. McKnelly KJ, Kreutzer AG, Howitz WJ, Haduong K, Yoo S, Hart C, Nowick JS. Effects of Familial Alzheimer's Disease Mutations on the Assembly of a β-Hairpin Peptide Derived from Aβ16-36. Biochemistry. 2022 Mar 15;61(6):446-454. Epub 2022 Feb 25 PubMed.
13. Kim M, Bezprozvanny I. Analysis of Non-Amyloidogenic Mutations in APP Supports Loss of Function Hypothesis of Alzheimer's Disease. Int J Mol Sci. 2023 Jan 20;24(3) PubMed.
14. Barrett PJ, Song Y, Van Horn WD, Hustedt EJ, Schafer JM, Hadziselimovic A, Beel AJ, Sanders CR. The amyloid precursor protein has a flexible transmembrane domain and binds cholesterol. Science. 2012 Jun 1;336(6085):1168-71. PubMed.
15. Nilsberth C, Westlind-Danielsson A, Eckman CB, Condron MM, Axelman K, Forsell C, Stenh C, Luthman J, Teplow DB, Younkin SG, Näslund J, Lannfelt L. The 'Arctic' APP mutation (E693G) causes Alzheimer's disease by enhanced Abeta protofibril formation. Nat Neurosci. 2001 Sep;4(9):887-93. PubMed.
16. Tsubuki S, Takaki Y, Saido TC. Dutch, Flemish, Italian, and Arctic mutations of APP and resistance of Abeta to physiologically relevant proteolytic degradation. Lancet. 2003 Jun 7;361(9373):1957-8. PubMed.

Further Reading