Oral Presentation Australasian RNA Biology and Biotechnology Association 2026 Conference

What’s in a Message? Long-Read RNA Sequencing Reveals Convergent RNA Regulatory and Axonal Pathways in Distal Hereditary Motor Neuropathy. (144934)

Madison D.V. Gonebale 1 2 , Cali Willet 3 , Amarinder Thind 3 , Mitchell O'Brien 3 , Michael Geaghan 3 , Georgina Samaha 3 , Steve Vucic 4 , Anthony N Cutrupi 1 2 , Marina L Kennerson 1 2 4
  1. ANZAC Research Institute, Concord, NSW, Australia
  2. School of Medical Sciences, University of Sydney, Faculty of Medicine, Camperdown, NSW, Australia
  3. Sydney Informatics Hub, Newtown, NSW, Australia
  4. Brain and Nerve Research Centre, Concord Hospital, Concord, NSW, Australia

We previously identified a disease associated complex structural variation causing distal hereditary motor neuropathy (DHMN1), which generates a novel gene-intergenic fusion transcript (UBE3C-IF) in induced pluripotent stem cell-derived patient spinal motor neurons (sMNs). While bulk short-read RNA sequencing confirmed UBE3C-IF expression, it failed to identify biologically meaningful downstream transcriptomic changes. Here, we apply cDNA PCR-amplified long-read RNA sequencing (lrRNA-seq) to define transcript-level alterations associated with DHMN1 to identify molecular mechanisms underlying the disease.  

 

RNA libraries for iPSC-derived sMNs (n=3 DHMN1 patients with segregating complex structural variation; n=3 controls) were prepared using the Oxford Nanopore Technologies cDNA-PCR Barcording Kit V14 and sequenced (PromethION P2S; R10.4.1 flow cells). Reads were aligned and quantified using our in-house pipeline (ORBiT) with transcript quantification performed using IsoQuant. Differential transcript expression (DTE) and usage (DTU) analyses were assessed using DESeq2 and DRIMSeq respectively (adjusted p<0.05, in addition to a log2fold change > ± 1 for DESeq2). We also developed a tool (GIFFL) to detect gene fusions created from gene-to-transcribed sequence derived from anywhere in the genome.

 

GIFFL validated the full-length UBE3C-IF transcript exclusively in DHMN1 sMNs. DTE analysis identified co-ordinated dysregulation of transcripts involved in RNA binding and processing (QKI and HARS1), together with reduced expression of transcripts involved with axon growth and synaptic transmission (DRAXIN, NALF2 and SYNPR). DTU analysis identified 70 genes with altered isoform usage, revealing convergence on RNA and transcriptional regulation (RBFOX1, RALYL, SNRPR and POLR2I) as well as axonal and synaptic pathways (VPS29, CD24, LSM5 and TUBA1A). Altered isoform usage was also observed in several non-coding RNAs, including the neuroprotective long non-coding RNA, ZNF667-AS1.  

 

Collectively these findings demonstrate that long-read transcriptomics reveals transcript- and isoform-level changes not detected by short-read RNA sequencing and links the DHMN1-associated structural variation and UBE3C-IF transcript to convergent disruption of RNA regulation and axonal/synapse biology. Despite the small cohort, the highly penetrant nature of DHMN1 and the shared segregating causal variant, provides a strong genetic framework for detecting disease relevant transcriptomic alterations. By incorporating best practice tools (ORBiT) and developing novel algorithms (GIFFL) this has provided a robust analytical pipeline for long-read transcriptomics and fusion transcript detection. Our study demonstrates the value of lrRNA-seq for uncovering disease mechanism and candidate molecular biomarkers for ultra-rare inherited neuropathies.