An atlas of amyloid aggregation: the impact of substitutions, insertions, deletions and truncations on amyloid beta fibril nucleation

Abstract

Multiplexed assays of variant effects (MAVEs) guide clinical variant interpretation and reveal disease mechanisms. To date, MAVEs have focussed on a single mutation type—amino acid (AA) substitutions—despite the diversity of coding variants that cause disease. Here we use Deep Indel Mutagenesis (DIM) to generate a comprehensive atlas of diverse variant effects for a disease protein, the amyloid beta (Aβ) peptide that aggregates in Alzheimer’s disease (AD) and is mutated in familial AD (fAD). The atlas identifies known fAD mutations and reveals that many variants beyond substitutions accelerate Aβ aggregation and are likely to be pathogenic. Truncations, substitutions, insertions, single- and internal multi-AA deletions differ in their propensity to enhance or impair aggregation, but likely pathogenic variants from all classes are highly enriched in the polar N-terminal region of Aβ. This comparative atlas highlights the importance of including diverse mutation types in MAVEs and provides important mechanistic insights into amyloid nucleation.

DIMPLE: deep insertion, deletion, and missense mutation libraries for exploring protein variation in evolution, disease, and biology

“…Substitutions are extremely context-dependent, with the impact of each depending on the physicochemical context in the structure. Consistent with other indel mutagenesis studies, we observed that deletions were in general more deleterious than insertions [1,6,30,31]. This effect becomes stronger with increasing length for both insertions and deletions as well (Additional file 3: Fig S8).…”

“…A prior indelscanning work provided evidence for this belief, as it revealed that most long indels are deleterious. Departures from this trend were also highly specific to the particular amyloid system, with idiosyncratic effects of large deletions being driven by exposure of a nucleating core and not generalizable to well folded proteins [1]. Based on this, we chose as default lengths 1, 2, and 3 amino acid-long indels, allowing us to capture the most relevant natural variation and observe any interesting length-dependent fitness effects while still maximizing sequencing capacity.…”

“…Each paradigm has produced fundamental insights into how nature produces life and what goes wrong in disease, but each often overlooks mutations beyond simple missense substitutions. Recent work has underscored how essential other types of mutations are to evolutionary novelty and adaptation, as well as their utility for understanding diseases and protein engineering [1][2][3][4][5][6]. In addition to missense mutations, we must consider frameshifts, recombination, splice variations, and insertions and deletions to evaluate how mutations change proteins.…”

“…These efforts have culminated in large-scale international efforts such as the Atlas of Variant Effects Alliance, with the goal of characterizing all variants circulating within human populations. However, while insertions and deletions (indels) make-up nearly 1 3 of disease-causing variants, to date only two pioneering DMS studies have included indels [1,6]. Of the two, one contains only single-codon deletions and insertions (but all possible insertions); the other comprehensively samples deletions and single codon insertions, but within a small (129 bp) gene that can be synthesized directly.…”

“…Because each variant is individually synthesized rather than randomly generated, OLS-based libraries are typically more complete, can include any variant type, and simpler to clone than PCR-based approaches. Indeed, the only two mutational scans to date that included indel variants were made with bespoke OLSbased approaches that may not generalize [1,6].…”