MINTIE: identifying novel structural and splice variants in transcriptomes using RNA-seq data

Cmero, Marek; Schmidt, Breon; Majewski, Ian J.; Ekert, Paul G.; Oshlack, Alicia; Davidson, N.

doi:10.1186/s13059-021-02507-8

Cited by 19 publications

(21 citation statements)

References 75 publications

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“…Splicing of the predicted 255 bp cryptic intron was not detected by RNA-seq, although a distinct drop in coverage over 78 bp neighbouring the 3’ UTR variant site was observed (Figure 2E,F) suggesting the formation of a 78 bp cryptic intron which would result in a secondary variant in the 3’ UTR of KLHL40 ; c.*151_*228del. MINTIE(15) alignment of the RNA-seq reads confirmed the 78 bp deletion (hg19; chr3:42733635-42733714) and indicated the presence of a new exon junction with a variant allele frequency of 0.94. This implies that 94% of the reads contained the cryptic 78 bp deletion.…”

Section: Resultsmentioning

confidence: 87%

“…RNA-seq data were visualised with the Integrative Genomics Viewer (IGV)(47). Data were also assembled and analysed using MINTIE(48), a reference-free RNA-seq analysis pipeline that maps reads de novo to identify novel structural and splice variants(15). Muscle RNA-seq FASTQ data from five additional non-nemaline myopathy patients were used as controls for MINTIE.…”

Section: Patient and Methodsmentioning

confidence: 99%

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A KLHL40 3’ UTR splice-altering variant causes milder NEM8, an under-appreciated disease mechanism

Dofash

Monahan

Servián‐Morilla

et al. 2022

Preprint

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Nemaline myopathy 8 (NEM8) is typically a severe autosomal recessive disorder associated with variants in the kelch-like family member 40 gene (KLHL40). Common features include fetal akinesia, fractures, contractures, dysphagia, respiratory failure, and neonatal death. Here, we describe a man in his 20s with relatively mild NEM8. He presented with hypotonia and bilateral femur fractures at birth, later developing bilateral Achilles' contractures, scoliosis, and elbow and knee contractures. He had walking difficulties throughout childhood and became wheelchair bound during adolescence after prolonged immobilisation. Muscle MRI during adolescence indicated prominent fat replacement in his pelvic girdle, posterior compartments of thighs, and vastus intermedius. Muscle biopsy revealed nemaline bodies and intranuclear rods. RNA sequencing and western blotting of patient skeletal muscle indicated significant reduction in KLHL40 mRNA and protein respectively. Using gene panel screening, exome sequencing and RNA sequencing, we identified compound heterozygous variants in KLHL40; a truncating 10.9 kb deletion in trans with a likely pathogenic variant (c.*152G>T) in the 3′ untranslated region (UTR). Computational tools SpliceAI and Introme predicted the c.*152G>T variant created a cryptic donor splice site. RNA-seq and in vitro analyses indicated that the c.*152G>T variant induces multiple de novo splicing events that likely provoke nonsense mediated decay of KLHL40 mRNA explaining the loss of mRNA expression and protein abundance in the patient. Analysis of 3′ UTR variants in ClinVar suggests SNPs that introduce aberrant 3′ UTR splicing may be underrecognised in Mendelian disease. We encourage consideration of this mechanism during variant curation.

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Section: Resultsmentioning

confidence: 87%

Section: Patient and Methodsmentioning

confidence: 99%

A KLHL40 3’ UTR splice-altering variant causes milder NEM8, an under-appreciated disease mechanism

Dofash

Monahan

Servián‐Morilla

et al. 2022

Preprint

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“…achieved de novo assembly of over a thousand TCGA samples ( 65 ), but this was restricted to unmapped reads, therefore missing most RNA variations. More recently, an assembly-first strategy was introduced to identify patient-specific abnormal transcripts under a ‘1 to n ’ experimental design ( 66 ). De novo read assembly produces longer contigs, which facilitates biological interpretation and is better suited to large structural variants.…”

Section: Discussionmentioning

confidence: 99%

The contribution of uncharted RNA sequences to tumor identity in lung adenocarcinoma

et al. 2022

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The identity of cancer cells is defined by the interplay between genetic, epigenetic transcriptional and post-transcriptional variation. A lot of this variation is present in RNA-seq data and can be captured at once using reference-free, k-mer analysis. An important issue with k-mer analysis, however, is the difficulty of distinguishing signal from noise. Here, we use two independent lung adenocarcinoma datasets to identify all reproducible events at the k-mer level, in a tumor versus normal setting. We find reproducible events in many different locations (introns, intergenic, repeats) and forms (spliced, polyadenylated, chimeric etc.). We systematically analyze events that are ignored in conventional transcriptomics and assess their value as biomarkers and for tumor classification, survival prediction, neoantigen prediction and correlation with the immune microenvironment. We find that unannotated lincRNAs, novel splice variants, endogenous HERV, Line1 and Alu repeats and bacterial RNAs each contribute to different, important aspects of tumor identity. We argue that differential RNA-seq analysis of tumor/normal sample collections would benefit from this type k-mer analysis to cast a wider net on important cancer-related events. The code is available at https://github.com/Transipedia/dekupl-lung-cancer-inter-cohort.

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“…93 Fusions involving intergenic/non-coding sequences that result in cryptic (pseudo) exon formation are by comparison rare -only being reported by a few studies. [93][94][95][96] Interestingly, the majority of genomic breakpoints in fusion genes are intergenic or intronic and are therefore not typically present in mRNA or protein-coding sequences 90,91,97 making detection of gene-intergenic fusions difficult. This may account for the predominance of classical gene-gene fusions over gene-intergenic fusions in the literature to date.…”

Section: Discussionmentioning

confidence: 99%

Novel gene-intergenic fusion involving ubiquitin E3 ligase UBE3C causes distal hereditary motor neuropathy: A new mechanism for motor neuron degeneration

Cutrupi

Narayanan

Perez-Siles

et al. 2022

Preprint

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Distal hereditary motor neuropathies (dHMNs) are a group of inherited diseases involving the progressive, length-dependent axonal degeneration of the lower motor neurons. There are currently 29 reported causative genes and 4 disease loci implicated in dHMN. Despite the high genetic heterogeneity, mutations in the known genes account for less than 20% of dHMN cases with the mutations identified predominantly being point mutations or indels. We have expanded the spectrum of dHMN mutations with the identification of a 1.35 Mb complex structural variation (SV) causing a form of autosomal dominant dHMN (DHMN1 OMIM %182906). Given the complex nature of SV mutations and the importance of studying pathogenic mechanisms in a neuronal setting, we generated a patient-derived DHMN1 motor neuron model harbouring the 1.35 Mb complex insertion. The DHMN1 complex insertion creates a duplicated copy of the first 10 exons of the ubiquitin-protein E3 ligase gene (UBE3C) and forms a novel gene-intergenic fusion sense transcript by incorporating a terminal pseudo-exon from intergenic sequence within the DHMN1 locus. The UBE3C intergenic fusion (UBE3C-IF) transcript does not undergo nonsense-mediated decay and results in a significant reduction of wild type full length UBE3C (UBE3C-WT) protein levels in DHMN1 iPSC-derived motor neurons. An engineered transgenic C. elegans model expressing the UBE3C-IF transcript in GABA-ergic motor neurons shows neuronal synaptic transmission deficits. Furthermore, the transgenic animals are susceptible to heat stress which may implicate defective protein homeostasis underlying DHMN1 pathogenesis. Identification of the novel UBE3C-IF gene-intergenic fusion transcript in motor neurons highlights a potential new disease mechanism underlying axonal and motor neuron degeneration. These complementary models serve as a powerful paradigm for studying the DHMN1 complex SV and an invaluable tool for defining therapeutic targets for DHMN1.

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MINTIE: identifying novel structural and splice variants in transcriptomes using RNA-seq data

Cited by 19 publications

References 75 publications

A KLHL40 3’ UTR splice-altering variant causes milder NEM8, an under-appreciated disease mechanism

A KLHL40 3’ UTR splice-altering variant causes milder NEM8, an under-appreciated disease mechanism

The contribution of uncharted RNA sequences to tumor identity in lung adenocarcinoma

Novel gene-intergenic fusion involving ubiquitin E3 ligase UBE3C causes distal hereditary motor neuropathy: A new mechanism for motor neuron degeneration

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