Evan Reid

RNA sequencing (RNA-Seq) is increasingly used alongside exome and genome sequencing to identify causal variants underlying rare Mendelian disorders. We present short-read RNA-Seq data from 5,412 individuals with a diverse range of rare disorders recruited to Genomics England’s 100,000 Genomes Project. We show that the proportion of genes from gene panels applied to different disorders which are well captured (transcripts per million (TPM) ≥ 5) from blood RNA varies widely, highlighting differences in applicability across disorder types. Using OUTRIDER and FRASER2 to identify gene expression and splicing outliers respectively, we identify at least one outlier event in a disorder relevant gene in 20% of the cohort. To prioritise likely diagnostic candidates, we apply multiple strategies including focussing on outlier events in known haploinsufficient genes (n=78), integrating outliers with structural variant calls (n=19), and using strategies integrating phenotypic presentation (Exomiser, n=39). We present a series of candidate diagnoses involving diverse variant types and disease mechanisms, demonstrating the broad utility of RNA-Seq in identifying and prioritising diagnostic candidates in individuals with a variety of different rare conditions and no known genetic diagnosis. Our findings demonstrate that blood-based RNA-Seq can deliver clinically relevant findings across a broad range of rare disorders.

Cholesterol is an essential molecule in all animals, it can be made by de novo synthesis and can be taken up from the diet. Inherited disorders of cholesterol synthesis, metabolism and transport lead to disease, often with neurological signs. However, such disorders tend to have non-specific symptoms and can be difficult to diagnose. In addition, there is no single diagnostic test applicable to multiple disorders of cholesterol synthesis, metabolism and transport which can be used to suggest or confirm a diagnosis, resulting in a delay in treatment, particularly in the case of unknown genetic variants. Here, we present the first version of a mass spectrometry sterolomic library to aid the diagnosis of manifold cholesterol-related inherited disorders of metabolism. The library was generated using technology based on simple derivatisation chemistry exploiting the Girard P hydrazine reagent and utilising electrospray ionisation mass spectrometry in the positive and negative modes. The library includes data for 13 autosomal recessive disorders and predicted data for a further 8 disorders.

Fission of transport tubules from early endosomes is required for endosomal sorting, but mechanisms of endosomal tubule fission (ETF) are incompletely understood. We show protrudin acts at ER-endosome contacts to promote ETF and endosome-to-Golgi traffic. Protrudin-mediated ETF required its ability to interact with ER-localised VAP proteins, endosomal phosphoinositides and KIF5. These properties also regulated the distance between protrudin and endosomal tubules. The defective ETF phenotype of increased endosomal tubulation in cells lacking protrudin was phenocopied by depletion of KIF5, but not FYCO1, a motor protein adaptor implicated in protrudin-dependent late endosome motility. It also required intact microtubules and dynein, consistent with a model where protrudin facilitates a tug-of-war between KIF5 and dynein to fission tubules. In addition to its direct role, protrudin links many other machineries involved in ETF, thus our findings elucidate how ETF is co-ordinated. These machineries are enriched for proteins implicated in hereditary motor neuron disorders, and protrudin or KIF5 depletion caused defective ETF in human neurons. Summary Protrudin binds ER-localised VAPs and endosomal phosphoinositides to form ER-endosome contacts that promote endosomal tubule fission and endosome-to-Golgi traffic. Protrudin recruits KIF5 to provide a FYCO1-independent force to fission endosomal tubules in neurons and non-polarised cells.

Solve-RD is a pan-European rare disease (RD) research program that aims to identify disease-causing genetic variants in previously undiagnosed RD families. We utilised 10-fold coverage HiFi long-read sequencing (LRS) for detecting causative structural variants (SVs), single nucleotide variants (SNVs), insertion-deletions (InDels), and short tandem repeat (STR) expansions in extensively studied RD families without clear molecular diagnoses. Our cohort includes 293 individuals from 114 genetically undiagnosed RD families selected by European Rare Disease Network (ERN) experts. Of these, 21 families were affected by so-called ‘unsolvable’ syndromes for which genetic causes remain unknown, and 93 families with at least one individual affected by a rare neurological, neuromuscular, or epilepsy disorder without genetic diagnosis despite extensive prior testing. Clinical interpretation and orthogonal validation of variants in known disease genes yielded thirteen novel genetic diagnoses due to de novo and rare inherited SNVs, InDels, SVs, and STR expansions. In an additional four families, we identified a candidate disease-causing SV affecting several genes including an MCF2 / FGF13 fusion and PSMA3 deletion. However, no common genetic cause was identified in any of the ‘unsolvable’ syndromes. Taken together, we found (likely) disease-causing genetic variants in 13.0% of previously unsolved families and additional candidate disease-causing SVs in another 4.3% of these families. In conclusion, our results demonstrate the added value of HiFi long-read genome sequencing in undiagnosed rare diseases.

Mutation of the ATL1 gene is one of the most common causes of hereditary spastic paraplegia (HSP), a group of genetic neurodegenerative conditions characterised by distal axonal degeneration of the corticospinal tract axons. Atlastin-1, the protein encoded by ATL1 , is one of three mammalian atlastins, which are homologous dynamin-like GTPases that control endoplasmic reticulum (ER) morphology by fusing tubules to form the three-way junctions that characterise ER networks. However, it is not clear whether atlastin-1 is required for correct ER morphology in human neurons and if so what the functional consequences of lack of atlastin-1 are. Using CRISPR-inhibition we generated human cortical neurons lacking atlastin-1. We demonstrate that ER morphology was altered in these neurons, with a reduced number of three-way junctions. Neurons lacking atlastin-1 had longer endosomal tubules, suggestive of defective tubule fission. This was accompanied by reduced lysosomal proteolytic capacity. As well as demonstrating that atlastin-1 is required for correct ER morphology in human neurons, our results indicate that lack of a classical ER-shaping protein such as atlastin-1 may cause altered endosomal tubulation and lysosomal proteolytic dysfunction. Furthermore, they strengthen the idea that defective lysosome function contributes to the pathogenesis of a broad group of HSPs, including those where the primary localisation of the protein involved is not at the endolysosomal system.

Purpose Despite significant progress in unravelling the genetic causes of neurodevelopmental disorders (NDDs), a substantial proportion of individuals with NDDs remain without a genetic diagnosis following microarray and/or exome sequencing. Here we aimed to assess the power of short-read genome sequencing (GS), complemented with long-read GS, to identify causal variants in NDD participants from the NIHR BioResource project. Methods Short-read GS was conducted on 692 individuals (489 affected and 203 unaffected relatives) from 465 families. Additionally, long-read GS was performed on five affected individuals who had structural variants (SVs) in technically challenging regions, complex SVs, or required distal variant phasing. Results Causal variants were identified in 36% affected individuals (177/489) and a further 26% (129/489) had a variant of uncertain significance, after multiple rounds of re-analysis. Among all reported variants, 88% (333/380) were SNVs/indels, and the remainder were structural variants (SVs), non-coding, and mitochondrial variants. Furthermore, long-read GS facilitated resolution of challenging SVs and invalidated variants of difficult interpretation from short-read GS. Conclusion This study demonstrates the value of short-read GS, complemented with long-reads, to investigate the genetic causes of NDDs. GS provides a comprehensive and unbiased method to identify all types of variants throughout the nuclear and mitochondrial genome in NDD individuals.

A subset of individuals diagnosed with cerebral palsy will have an underlying genetic diagnosis. Previously, a missense variant in GAD1 was described as a candidate mutation in a single family diagnosed with autosomal recessive spastic cerebral palsy-1 (CPSQ1; OMIM 603513). Following the ascertainment of a further branch of the CPSQ1 kindred, we found that the previously reported GAD1 variant did not segregate with the neurological disease phenotype in the recently ascertained branch of the kindred. Following genetic linkage studies to map autozygous regions and whole-exome sequencing, a missense variant (c.527 T > C; p. Leu176Pro, rs773333490) in the HPDL gene was detected and found to segregate with disease status in both branches of the kindred. HPDL encodes a 371-amino acid protein (4-Hydroxyphenylpyruvate Dioxygenase Like) that localizes to mitochondria but whose function is uncertain. Recently, biallelic loss of function variants and missense substitution-causing variants in HPDL were reported to cause a childhood onset progressive spastic movement disorder with a variable presentation. These findings suggest that HPDL-related neurological disease may mimic spastic cerebral palsy and that GAD1 should not be included in diagnostic gene panels for inherited cerebral palsy.