Investigating short structural variants within FUS, RAB27B and TARDBP for associations with sporadic amyotrophic lateral sclerosis

Caitlyn Vicars

Over the past decade, 90% of amyotrophic lateral sclerosis (ALS) clinical trials have failed. Furthermore, 90% of sporadic cases have unknown genetic cause. Short structural variants (sSVs) are repetitive genomic regions implicated in complex diseases, with the potential to be utilised for clinical trial enrichment. The purpose of this study was to investigate sporadic ALS (sALS) missing heritability via interrogating novel sSVs, and to determine their potential as genetic biomarkers. This project aimed to develop assays for characterisation and high-throughput genotyping of sSVs within ALS-linked genes FUS and TARDBP, and the novel gene, RAB27B, to perform case-control analyses. Three loci; FUS rs34242298, TARDBP rs34839760 and RAB27B rs3060044, were selected using an sSV evaluation algorithm. Polymorphic nature was characterised via polymerase chain reaction-based assays and Sanger sequencing. High-throughput genotyping of sALS patients and healthy controls was undertaken via capillary electrophoresis, followed by case-control analyses in RStudio to identify statistically significant (p<0.05) associations with disease risk and/or site-of-onset. Reduced sALS risk was associated with Short rs3060044 alleles with ≤17GT repeats (p<0.05), driven by 16GT allele carriage. Increased disease risk correlated with carriage of a Long allele (>17GT repeats) (p<0.05), driven by the 21GT allele (p=0.00625). Analysis of rs34839760 revealed the 20A allele was associated with bulbar-onset phenotype (p<0.05), while rs34242298 was not associated with sALS. This exploratory study reinforces the requirement for sSV investigation in complex diseases. Two novel sALS genetic loci were identified; rs3060044 and rs34839760, which enhance our understanding of the aetiology and pathomechanisms underlying sALS. This data is the first to implicate RAB27B as an sALS gene, presenting a tantalising direction for future sALS exploration. Replication studies and functional investigations are warranted to explore the role of each sSV in sALS pathology. Ultimately, these genomic markers could be useful as patient stratification tools, which may enable the identification of responders to precision therapeutics.

Investigating short structural variants within FUS, RAB27B and TARDBP for associations with sporadic amyotrophic lateral sclerosis

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