The Landscape of SNCA Transcripts Across Synucleinopathies: New Insights From Long Reads Sequencing Analysis

Abstract

Dysregulation of alpha-synuclein expression has been implicated in the pathogenesis of synucleinopathies, in particular Parkinson’s Disease (PD) and Dementia with Lewy bodies (DLB). Previous studies have shown that the alternatively spliced isoforms of the SNCA gene are differentially expressed in different parts of the brain for PD and DLB patients. Similarly, SNCA isoforms with skipped exons can have a functional impact on the protein domains. The large intronic region of the SNCA gene was also shown to harbor structural variants that affect transcriptional levels. Here, we apply the first study of using long read sequencing with targeted capture of both the gDNA and cDNA of the SNCA gene in brain tissues of PD, DLB, and control samples using the PacBio Sequel system. The targeted full-length cDNA (Iso-Seq) data confirmed complex usage of known alternative start sites and variable 3′ UTR lengths, as well as novel 5′ starts and 3′ ends not previously described. The targeted gDNA data allowed phasing of up to 81% of the ~114 kb SNCA region, with the longest phased block exceeding 54 kb. We demonstrate that long gDNA and cDNA reads have the potential to reveal long-range information not previously accessible using traditional sequencing methods. This approach has a potential impact in studying disease risk genes such as SNCA, providing new insights into the genetic etiologies, including perturbations to the landscape the gene transcripts, of human complex diseases such as synucleinopathies.

Fine‐Mapping of SNCA in Rapid Eye Movement Sleep Behavior Disorder and Overt Synucleinopathies

“…In contrast, a variant in the 3′ end of the intron‐4 enhancer region rs356168‐A has been associated with increased levels of α‐synuclein in human induced pluripotent stem cells and is in LD with the top signal for PD rs356182‐A ( R 2 = 0.50, D′ = 0.91). Finally, the RBD 5′ variant rs10005233 has been linked to novel alternative 3′‐end SNCA isoforms (PB.1016.383, PB.1016.384), associated with a truncated open reading frame prediction . These findings suggest potential functional effects of some of these variants, but they need to be further replicated and studied.…”

Next-Generation RNA-Sequencing of Serum Small Extracellular Vesicles Discovers Potential Diagnostic Biomarkers for Dementia With Lewy Bodies

“…DNA repair deficits and consequent increased levels of DNA double strand breaks are associated with Lewy body pathology and neurodegeneration in mice models and postmortem human DLB brains (47). Moreover, transcriptional RNA processing such as RNA splicing contributes to α-synuclein aggregation (48). We have presented a dysfunctional molecular network involving several identified DEGs that can affect DNA repair and RNA post-transcriptional modification.…”

Accurate spliced alignment of long RNA sequencing reads

“…Long-read sequencing technologies can sequence transcripts from end to end, providing the full isoform structure and therefore offer accurate isoform detection and quantification. Such protocols have opened up the possibility to investigate the isoform landscape for genes with multiple gene copies ( Sahlin et al , 2018 ) and complex splicing patterns ( Tseng et al , 2019 ), as well as to accurately decipher alleles ( Tilgner et al , 2014 ) and cell-specific ( Gupta et al , 2018 ) isoforms. However, the long-read technologies also offer new algorithmic challenges because of the higher error rate and longer sequencing length which makes most short-read alignment algorithms unsuitable for long-read splice alignment ( Križanović et al , 2018 ).…”