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Uveal melanoma (UM) is the most common primary intraocular malignancy in adults. Owing to a lack of effective treatments, patients with metastatic disease have a median survival time of 6-12 months. We recently demonstrated that the Survival Associated Mitochondrial Melanoma Specific Oncogenic Non-coding RNA (SAMMSON) is essential for UM cell survival and that antisense oligonucleotide (ASO)-mediated silencing of SAMMSON impaired cell viability and tumor growth in vitro and in vivo . By screening a library of 2911 clinical stage compounds, we identified the mammalian target of rapamycin (mTOR) inhibitor GDC-0349 to synergize with SAMMSON inhibition in UM. Mechanistic studies revealed that mTOR inhibition enhanced uptake and reduced lysosomal accumulation of lipid complexed SAMMSON ASOs, improving SAMMSON knockdown and further decreasing UM cell viability. We found mTOR inhibition to also enhance target knockdown in other cancer cell lines as well as normal cells when combined with lipid nanoparticle complexed or encapsulated ASOs or small interfering RNAs (siRNAs). Our results are relevant to nucleic acid treatment in general and highlight the potential of mTOR inhibition to enhance ASO and siRNA-mediated target knockdown.

There is increasing evidence that diseases caused by dysfunctional mitochondria (MD) are associated with autism spectrum disorder (ASD). A comprehensive meta-analysis showed that developmental regression was reported in half of the children with ASD and mitochondrial dysfunction which is much higher than in the general population of ASD. The aim of the present exploratory study was to determine lactate concentrations in urine of children with ASD, as a non-invasive large-scale screening method for metabolic abnormalities including mitochondrial dysfunction and its possible association with regression. First, clinical characteristics of MD were examined in 99 children (3-11 years) with ASD. Second, clinical characteristics of MD, severity of ASD and reported regression were compared between children with the 20% lowest lactate concentrations and those with the 20% highest lactate concentrations in urine. Third, clinical characteristics of MD and lactate concentration in urine were compared in children with (n = 37) and without (n = 62) reported regression. An association of urine lactate concentrations with mitochondrial dysfunction and regression could not be demonstrated in our large ASD cohort. However, since ASD children were reported by their parents to show a broad range of phenotypic characteristics of MD (e.g., gastro-intestinal and respiratory impairments), and lactate concentrations in urine are not always increased in individuals with MD, the presence of milder mitochondrial dysfunction cannot be excluded. Development of alternative biomarkers and their implementation in prospective studies following developmental trajectories of infants at elevated likelihood for ASD will be needed in the future to further unravel the association of ASD with mitochondrial dysfunction and eventually improve early detection., Competing Interests: The authors have declared that no competing interests exist.

Background and Purpose: Ataxia and cough are rare features in hereditary sensory and autonomic neuropathies (HSAN), a group of diseases of mostly unknown genetic cause. Biallelic repeat expansions in RFC1 are associated with cerebellar ataxia, neuropathy, and vestibular areflexia syndrome (CANVAS). This study aimed to investigate the prevalence of RFC1 repeat expansions in a cohort of HSAN patients., Methods: After unremarkable whole-exome sequencing (WES) analysis, we performed repeat-primed PCR to detect intronic RFC1 expansions in 12 HSAN families, who all presented with chronic cough., Results: In these patients, 75% carried biallelic expansions of the pathogenic AAGGG motif. Compared with RFC1-/- cases, RFC1+/+ cases presented more consistently with positive sensory and autonomic symptoms. Afferent ataxia was more severe in the RFC1+/+ cohort and cerebellar ataxia was a common feature (21%)., Conclusions: We demonstrate that RFC1 is a frequent cause of (WES-negative) HSAN with chronic cough and ataxia. The diagnostic yield of RFC1 repeat-primed PCR was surprisingly high, given that HSAN is genetically poorly understood. This combination of HSAN, ataxia, and chronic cough symptoms represents a new nosological entity within the neuropathy-ataxia spectrum., (© 2022 European Academy of Neurology.)

Background: In order to facilitate the diagnostic process for adult patients suffering from a rare disease, the Undiagnosed Disease Program (UD-PrOZA) was founded in 2015 at the Ghent University Hospital in Belgium. In this study we report the five-year results of our multidisciplinary approach in rare disease diagnostics., Methods: Patients referred by a healthcare provider, in which an underlying rare disease is likely, qualify for a UD-PrOZA evaluation. UD-PrOZA uses a multidisciplinary clinical approach combined with state-of-the-art genomic technologies in close collaboration with research facilities to diagnose patients., Results: Between 2015 and 2020, 692 patients (94% adults) were referred of which 329 (48%) were accepted for evaluation. In 18% (60 of 329) of the cases a definite diagnosis was made. 88% (53 of 60) of the established diagnoses had a genetic origin. 65% (39 of 60) of the genetic diagnoses were made through whole exome sequencing (WES). The mean time interval between symptom-onset and diagnosis was 19 years. Key observations included novel genotype-phenotype correlations, new variants in known disease genes and the identification of three new disease genes. In 13% (7 of 53), identifying the molecular cause was associated with therapeutic recommendations and in 88% (53 of 60), gene specific genetic counseling was made possible. Actionable secondary findings were reported in 7% (12 of 177) of the patients in which WES was performed., Conclusion: UD-PrOZA offers an innovative interdisciplinary platform to diagnose rare diseases in adults with previously unexplained medical problems and to facilitate translational research., (© 2022. The Author(s).)

Background: Lack of functional evidence hampers variant interpretation, leaving a large proportion of individuals with a suspected Mendelian disorder without genetic diagnosis after whole genome or whole exome sequencing (WES). Research studies advocate to further sequence transcriptomes to directly and systematically probe gene expression defects. However, collection of additional biopsies and establishment of lab workflows, analytical pipelines, and defined concepts in clinical interpretation of aberrant gene expression are still needed for adopting RNA sequencing (RNA-seq) in routine diagnostics., Methods: We implemented an automated RNA-seq protocol and a computational workflow with which we analyzed skin fibroblasts of 303 individuals with a suspected mitochondrial disease that previously underwent WES. We also assessed through simulations how aberrant expression and mono-allelic expression tests depend on RNA-seq coverage., Results: We detected on average 12,500 genes per sample including around 60% of all disease genes-a coverage substantially higher than with whole blood, supporting the use of skin biopsies. We prioritized genes demonstrating aberrant expression, aberrant splicing, or mono-allelic expression. The pipeline required less than 1 week from sample preparation to result reporting and provided a median of eight disease-associated genes per patient for inspection. A genetic diagnosis was established for 16% of the 205 WES-inconclusive cases. Detection of aberrant expression was a major contributor to diagnosis including instances of 50% reduction, which, together with mono-allelic expression, allowed for the diagnosis of dominant disorders caused by haploinsufficiency. Moreover, calling aberrant splicing and variants from RNA-seq data enabled detecting and validating splice-disrupting variants, of which the majority fell outside WES-covered regions., Conclusion: Together, these results show that streamlined experimental and computational processes can accelerate the implementation of RNA-seq in routine diagnostics., (© 2022. The Author(s).)

Facioscapulohumeral muscular dystrophy (FSHD) is an inherited myopathy clinically characterized by weakness in the facial, shoulder girdle and upper a muscles. FSHD is caused by chromatin relaxation of the D4Z4 macrosatellite repeat, mostly by a repeat contraction, facilitating ectopic expression of DUX4 in skeletal muscle. Genetic diagnosis for FSHD is generally based on the sizing and haplotyping of the D4Z4 repeat on chromosome 4 by Southern blotting (SB), molecular combing or single-molecule optical mapping, which is usually straight forward but can be complicated by atypical rearrangements of the D4Z4 repeat. One of these rearrangements is a D4Z4 proximally extended deletion (DPED) allele, where not only the D4Z4 repeat is partially deleted, but also sequences immediately proximal to the repeat are lost, which can impede accurate diagnosis in all genetic methods. Previously, we identified several DPED alleles in FSHD and estimated the size of the proximal deletions by a complex pulsed-field gel electrophoresis and SB strategy. Here, using the next-generation sequencing, we have defined the breakpoint junctions of these DPED alleles at the base pair resolution in 12 FSHD families and 4 control individuals facilitating a PCR-based diagnosis of these DPED alleles. Our resultsshow that half of the DPED alleles are derivates of an ancient founder allele. For some DPED alleles, we found that genetic elements are deleted such as DUX4c, FRG2, DBE-T and myogenic enhancers necessitating re-evaluation of their role in FSHD pathogenesis., (© The Author(s) 2021. Published by Oxford University Press. All rights reserved. For Permissions, please email: journals.permissions@oup.com.)

Long non-coding RNAs (lncRNAs) can exhibit cell-type and cancer-type specific expression profiles, making them highly attractive as therapeutic targets. Pan-cancer RNA sequencing data revealed broad expression of the SAMMSON lncRNA in uveal melanoma (UM), the most common primary intraocular malignancy in adults. Currently, there are no effective treatments for UM patients with metastatic disease, resulting in a median survival time of 6-12 months. We aimed to investigate the therapeutic potential of SAMMSON inhibition in UM. Antisense oligonucleotide (ASO)-mediated SAMMSON inhibition impaired the growth and viability of a genetically diverse panel of uveal melanoma cell lines. These effects were accompanied by an induction of apoptosis and were recapitulated in two uveal melanoma patient derived xenograft (PDX) models through subcutaneous ASO delivery. SAMMSON pulldown revealed several candidate interaction partners, including various proteins involved in mitochondrial translation. Consequently, inhibition of SAMMSON impaired global, mitochondrial and cytosolic protein translation levels and mitochondrial function in uveal melanoma cells. The present study demonstrates that SAMMSON expression is essential for uveal melanoma cell survival. ASO-mediated silencing of SAMMSON may provide an effective treatment strategy to treat primary and metastatic uveal melanoma patients., (© 2021. The Author(s).)

Deficiency of the serine hydrolase prolyl endopeptidase-like (PREPL) causes a recessive metabolic disorder characterized by neonatal hypotonia, feeding difficulties, and growth hormone deficiency. The pathophysiology of PREPL deficiency and the physiological substrates of PREPL remain largely unknown. In this study, we connect PREPL with mitochondrial gene expression and oxidative phosphorylation by analyzing its protein interactors. We demonstrate that the long PREPL L isoform localizes to mitochondria, whereas PREPL S remains cytosolic. Prepl KO mice showed reduced mitochondrial complex activities and disrupted mitochondrial gene expression. Furthermore, mitochondrial ultrastructure was abnormal in a PREPL-deficient patient and Prepl KO mice. In addition, we reveal that PREPL has (thio)esterase activity and inhibition of PREPL by Palmostatin M suggests a depalmitoylating function. We subsequently determined the crystal structure of PREPL, thereby providing insight into the mechanism of action. Taken together, PREPL is a (thio)esterase rather than a peptidase and PREPL L is involved in mitochondrial homeostasis., Competing Interests: The authors declare no competing interests., (© 2021 The Author(s).)