mTOR Inhibition Enhances Delivery and Activity of Antisense Oligonucleotides in Uveal Melanoma Cells.

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.

Case report: Thirty-year progression of an EMPF1 encephalopathy due to defective mitochondrial and peroxisomal fission caused by a novel de novo heterozygous DNM1L variant.

Mutations in DNM1L (DRP1), which encode a key player of mitochondrial and peroxisomal fission, have been reported in patients with the variable phenotypic spectrum, ranging from non-syndromic optic atrophy to lethal infantile encephalopathy. Here, we report a case of an adult female patient presenting with a complex neurological phenotype that associates axonal sensory neuropathy, spasticity, optic atrophy, dysarthria, dysphasia, dystonia, and ataxia, worsening with aging. Whole-exome sequencing revealed a heterozygous de novo variant in the GTPase domain of DNM1L [NM_001278464.1: c.176C>A p.(Thr59Asn)] making her the oldest patient suffering from encephalopathy due to defective mitochondrial and peroxisomal fission-1. In silico analysis suggested a protein destabilization effect of the variant Thr59Asn. Unexpectedly, Western blotting disclosed profound decrease of DNM1L expression, probably related to the degradation of DNM1L complexes. A detailed description of mitochondrial and peroxisomal anomalies in transmission electron and 3D fluorescence microscopy studies confirmed the exceptional phenotype of this patient.

Urine lactate concentration as a non-invasive screener for metabolic abnormalities: Findings in children with autism spectrum disorder and regression.

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.

Shortcutting the diagnostic odyssey: the multidisciplinary Program for Undiagnosed Rare Diseases in adults (UD-PrOZA).

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.

Clinical implementation of RNA sequencing for Mendelian disease diagnostics.

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.

RFC1 repeat expansions: A recurrent cause of sensory and autonomic neuropathy with cough and ataxia.

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.

The long non-coding RNA SAMMSON is essential for uveal melanoma cell survival.

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.

High-resolution breakpoint junction mapping of proximally extended D4Z4 deletions in FSHD1 reveals evidence for a founder effect.

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.

Prolyl endopeptidase-like is a (thio)esterase involved in mitochondrial respiratory chain function.

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 PREPLL isoform localizes to mitochondria, whereas PREPLS 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 PREPLL is involved in mitochondrial homeostasis.

Phenotypes and genotypes in non-consanguineous and consanguineous primary microcephaly: High incidence of epilepsy.

BACKGROUND: Primary microcephaly (PM) is defined as a significant reduction in occipitofrontal circumference (OFC) of prenatal onset. Clinical and genetic heterogeneity of PM represents a diagnostic challenge. METHODS: We performed detailed phenotypic and genomic analyses in a large cohort (n = 169) of patients referred for PM and could establish a molecular diagnosis in 38 patients. RESULTS: Pathogenic variants in ASPM and WDR62 were the most frequent causes in non-consanguineous patients in our cohort. In consanguineous patients, microarray and targeted gene panel analyses reached a diagnostic yield of 67%, which contrasts with a much lower rate in non-consanguineous patients (9%). Our series includes 11 novel pathogenic variants and we identify novel candidate genes including IGF2BP3 and DNAH2. We confirm the progression of microcephaly over time in affected children. Epilepsy was an important associated feature in our PM cohort, affecting 34% of patients with a molecular confirmation of the PM diagnosis, with various degrees of severity and seizure types. CONCLUSION: Our findings will help to prioritize genomic investigations, accelerate molecular diagnoses, and improve the management of PM patients.

Phenotypic spectrum of the RBM10-mediated intellectual disability and congenital malformation syndrome beyond classic TARP syndrome features.

Pathogenic variants in the RBM10 gene cause a rare X-linked disorder described as TARP (Talipes equinovarus, Atrial septal defect, Robin sequence, and Persistent left vena cava superior) syndrome. We report two novel patients with truncating RBM10 variants in view of the literature, presenting a total of 26 patients from 15 unrelated families. Our results illustrate the highly pleiotropic nature of RBM10 pathogenic variants, beyond the classic TARP syndrome features. Major clinical characteristics include severe developmental delay, failure to thrive, brain malformations, neurological symptoms, respiratory issues, and facial dysmorphism. Minor features are growth retardation, cardiac, gastrointestinal, limb, and skeletal abnormalities. Additional recurrent features include genital and renal abnormalities as well as hearing and visual impairment. Thus, RBM10 loss of function variants typically cause an intellectual disability and congenital malformation syndrome that requires assessment of multiple organ systems at diagnosis and for which provided clinical features might simplify diagnostic assessment. Furthermore, evidence for an RBM10-related genotype-phenotype correlation is emerging, which can be important for prognosis.

Mild myopathic phenotype in a patient with homozygous c.416C > T mutation in TK2 gene.

The mitochondrial DNA depletion syndrome (MDDS) is characterized by extensive phenotypic variability and is due to nuclear gene mutations resulting in reduced mtDNA copy number. Thymidine kinase 2 (TK2) mutations are well known to be associated with MDDS. Few severely affected cases carrying the c.416C > T mutation in TK2 gene have been described so far. We describe the case of a 14months boy with the aforementioned TK2 gene pathogenic mutation at a homozygous state, presenting with a mild clinical phenotype. In addition to severe mitochondrial pathology on muscle biopsy, there was also histochemical evidence of adenylate deaminase deficiency. Overall, this report serves to further expand the clinical spectrum of TK2 mutations associated with MDDS.

Functional characterization of novel MFSD8 pathogenic variants anticipates neurological involvement in juvenile isolated maculopathy.

Biallelic MFSD8 variants are an established cause of severe late-infantile subtype of neuronal ceroid lipofuscinosis (v-LINCL), a severe lysosomal storage disorder, but have also been associated with nonsyndromic adult-onset maculopathy. Here, we functionally characterized two novel MFSD8 variants found in a child with juvenile isolated maculopathy, in order to establish a refined prognosis. ABCA4 locus resequencing was followed by the analysis of other inherited retinal disease genes by whole exome sequencing (WES). Minigene assays and cDNA sequencing were used to assess the effect of a novel MFSD8 splice variant. MFSD8 expression was quantified with qPCR and overexpression studies were analyzed by immunoblotting. Transmission electron microscopy (TEM) was performed on a skin biopsy and ophthalmological and neurological re-examinations were conducted. WES revealed two novel MFSD8 variants: c.[590del];[439+3A>C] p.[Gly197Valfs*2];[Ile67Glufs*3]. Characterization of the c.439+3A>C variant via splice assays showed exon-skipping (p.Ile67Glufs*3), while overexpression studies of the corresponding protein indicated expression of a truncated polypeptide. In addition, a significantly reduced MFSD8 RNA expression was noted in patient's lymphocytes. TEM of a skin biopsy revealed typical v-LINCL lipopigment inclusions while neurological imaging of the proband displayed subtle cerebellar atrophy. Functional characterization demonstrated the pathogenicity of two novel MFSD8 variants, found in a child with an initial diagnosis of juvenile isolated maculopathy but likely evolving to v-LINCL with a protracted disease course. Our study allowed a refined neurological prognosis in the proband and expands the natural history of MFSD8-associated disease.

Nanoscopic X-ray imaging and quantification of the iron cellular architecture within single fibroblasts of Friedreich's ataxia patients.

Friedreich's ataxia (FRDA) is a neurodegenerative disease characterized by an increase in intracytoplasmic iron concentration. Here the nanoscale iron distribution within single fibroblasts from FRDA patients was investigated using synchrotron-radiation-based nanoscopic X-ray fluorescence and X-ray in-line holography at the ID16A nano-imaging beamline of the ESRF. This unique probe was deployed to uncover the iron cellular two-dimensional architecture of freeze-dried FRDA fibroblasts. An unsurpassed absolute detection capability of 180 iron atoms within a 30 nm × 50 nm nanoscopic X-ray beam footprint was obtained using state-of-the-art X-ray focusing optics and a large-solid-angle detection system. Various micrometre-sized iron-rich organelles could be revealed for the first time, tentatively identified as endoplasmic reticulum, mitochondria and lysosomes. Also a multitude of nanoscopic iron hot-spots were observed in the cytosol, interpreted as chaperoned iron within the fibroblast's labile iron pool. These observations enable new hypotheses on the storage and trafficking of iron in the cell and ultimately to a better understanding of iron-storage diseases such as Friedreich's ataxia.

Diagnostic utility of small fiber analysis in skin biopsies from children with chronic pain.

INTRODUCTION: Small fiber neuropathies (SFN) are associated with a reduction in quality of life. In adults, epidermal nerve fiber density (END) analysis is recommended for the diagnosis of SFN. In children, END assessment is not often performed. We analyzed small nerve fiber innervation to elucidate the potential diagnostic role of skin biopsies in young patients with pain. METHODS: Epidermal nerve fiber density and sudomotor neurite density (SND) were assessed in skin biopsies from 26 patients aged 7 to 20 years (15 female patients) with unexplained chronic pain. The results were compared with clinical data. RESULTS: Epidermal nerve fiber density was abnormal in 50% and borderline in 35% of patients. An underlying medical condition was found in 42% of patients, including metabolic, autoimmune, and genetic disorders. DISCUSSION: Reduction of epidermal nerve fibers can be associated with treatable conditions. Therefore, the analysis of END in children with pain may help to uncover a possible cause and guide potential treatment options.

Characteristics, Early Development and Outcome of Parent-Reported Regression in Autism Spectrum Disorder.

This study explored regression patterns in 100 children with ASD (3-11 years) using several approaches to enhance the validity of retrospective parent report. Both early development and outcome were examined in regression groups defined by 36 months age cut-off and two underlying empirical patterns based on type and onset age. Results over regression groups were generally consistent. During early development, children with regression showed a similar amount of social atypicalities and stereotyped behaviour as compared to children without regression. However, parents indicated less communication skills which could be a valuable predictor of regression. Development after regression was characterised by early language delay and more restricted and repetitive behaviour. The findings provide insight into the diagnosis and prognosis of regression in ASD.

Recurrent arterial ischemic stroke with good response to mycophenolate mofetil.

BACKGROUND: Arterial ischemic stroke is rare in childhood. Often, the diagnosis is made after considerable delay. A thorough workup to pinpoint the underlying etiology is necessary, as a correct diagnosis is the determining factor in treatment decision. In case of primary angiitis of the central nervous system, treatment with corticosteroids and immunosuppressive agents is indicated. CASE STUDY: We described an eleven-year-old boy who presented at the age of six years with left hemiparesis and hemianopia. Cerebral imaging showed acute ischemia in the right posterior cerebral artery territory. Extensive workup was negative. In the following eight months, he had recurrent strokes on three separate occasions due to progressive arteriopathy involving multiple large- and medium-sized vessels. A presumed diagnosis of primary angiitis of the central nervous system was made. Pulse intravenous methylprednisolone therapy was started followed by oral prednisolone. After the fourth stroke, a six-month treatment with cyclophosphamide was given which was followed by maintenance treatment with azathioprine. Shortly after cessation of corticosteroids and cyclophosphamide the subject relapsed. Cyclophosphamide was restarted in combination with corticosteroids and subsequently replaced by mycophenolate mofetil. Under mycophenolate mofetil maintenance treatment combined with low-dose corticosteroids, the patient achieved disease control with a relapse-free period of more than four years. CONCLUSION: A guideline for current treatment of relapsing central nervous system angiitis in childhood is missing in the literature. We describe a subject with multiple relapses despite treatment with corticosteroids and immunosuppressive agents, and stabilization of his clinical condition and of the radiological signs under mycophenolate mofetil treatment.

Leigh syndrome followed by parkinsonism in an adult with homozygous c.626C>T mutation in MTFMT.

OBJECTIVE: To report the clinical, radiologic, biochemical, and molecular characteristics in a 46-year-old participant with adult-onset Leigh syndrome (LS), followed by parkinsonism. METHODS: Case description with diagnostic workup included blood and CSF analysis, skeletal muscle investigations, blue native polyacrylamide gel electrophoresis, whole exome sequencing targeting nuclear genes involved in mitochondrial transcription and translation, cerebral MRI, 123I-FP-CIT brain single-photon emission computed tomography (SPECT), and C-11 raclopride positron emission tomography (PET). RESULTS: The participant was found to have a defect in the oxidative phosphorylation caused by a c.626C>T mutation in the gene coding for mitochondrial methionyl-tRNA formyltransferase (MTFMT), which is a pathogenic mutation affecting intramitochondrial protein translation. The proband had a normal concentration of lactate in blood and no abnormal microscopic findings in skeletal muscle. Cerebral MRI showed bilateral lesions in the striatum, mesencephalon, pons, and medial thalamus. Lactate concentration in CSF was increased. FP-CIT SPECT and C-11 raclopride PET demonstrated a defect in the dopaminergic system. CONCLUSIONS: We report on a case with adult-onset LS related to a MTFMT mutation. Two years after the onset of symptoms of LS, the proband developed a parkinson-like disease. The c.626C>T mutation is the most common pathogenic mutation found in 22 patients reported earlier in the literature with a defect in MTFMT. The age of the previously reported cases varied between 14 months and 24 years. Our report expands the phenotypical spectrum of MTFMT-related neurologic disease and provides clinical evidence for involvement of MTFMT in extrapyramidal syndromes.