New mutational mechanisms in ABCA7 influence Alzheimer's disease

Ondertitel:a third generation sequencing approach

Dementia is the fifth leading cause of death in the world. To find a therapy, we need a better understanding of the underlying disease causes, to which we aimed to contribute by studying the genetics of Alzheimer’s disease (AD), the predominant form of dementia. In particular, we focused our work on the identification and characterization of complex genetic variants in the ATP Binding Cassette Subfamily A Member 7 (ABCA7) gene. Our research group was one of the first to demonstrate a strong AD risk increasing effect of rare ABCA7 premature termination codon (PTC) variants. We first expanded on this striking finding by next-generation sequencing (NGS) analysis of coding ABCA7 variants in early-onset AD patients. We identified novel PTC variants and observed an overall fivefold PTC enrichment in patients. Because of incomplete disease penetrance and a wide onset age range, we characterized these PTC mutations on the transcript level by long-read sequencing on a MinION sequencer. We detected frequent transcript alterations which can rescue a deleterious PTC effect and are a possible target for therapeutic interventions. NGS, however, has limitations. By analysis of DNA sequences that are disregarded by conventional NGS processing, we were able to identify a highly polymorphic intronic ABCA7 variable number tandem repeat (VNTR). Subsequent Southern blotting revealed that VNTR expansions had a striking fourfold AD risk increasing effect. In downstream analyses we demonstrated that increasing VNTR lengths correlated with reduced ABCA7 expression and increased alternative splicing of a defunct ABCA7 isoform. Furthermore, we observed reduced amyloid ? levels in cerebrospinal fluid of AD patients carrying long VNTR lengths. Subsequently we aimed to develop a novel genotyping method based on long-read sequencing to enable improved characterization of this ABCA7 VNTR and other tandem repeats. We made use of the recently released high-throughput PromethION sequencing platform, and were the first to establish human whole genome long-read sequencing using only a single flow cell per individual. Finally, we developed “NanoSatellite”, a dynamic time warping-based tandem repeat calling algorithm, which demonstrated accurate length estimation for all ABCA7 VNTR lengths, including clinically relevant expansions, and detection of tandem repeat motif interruptions. In conclusion, we contributed to the establishment of ABCA7 as a strong risk factor of AD, which opens opportunities for novel drug targets and smarter AD clinical trials. In the process, we established novel methods, which will hopefully enable other researchers to study complex alterations in our genome and transcriptome.

Jaar van publicatie:2019

Trefwoorden:Doctoral thesis

Toegankelijkheid:Closed