BACKGROUND: Hearing loss (HL) is one of the most common congenital conditions and exhibits substantial clinical and genetic heterogeneity. More than 150 genes are associated with non-syndromic hearing loss (NSHL), while over 600 genes are linked to syndromic hearing loss (SHL). Importantly, the absence of additional clinical symptoms at the time of diagnosis does not necessarily exclude SHL. An increasing number of functionally disruptive variants in a growing number of genes have been shown to initially present as isolated HL, only later revealing syndromic features. METHODS: We analyzed clinical data from 111 patients across 102 unrelated families, selected from over 600 individuals negative for GJB2 and STRC variants. Molecular inversion probe panel, exome, or genome sequencing was performed, and patients were retrospectively divided into three subgroups following variant interpretation. Molecular docking was performed on select non-synonymous substitutions. RESULTS: Subgroup 1 included 30 patients with variants in neurodevelopmental disorder (NDD)-associated genes. HL was the first clinical manifestation in 80% of patients, with it being the sole first symptom in half. Subgroup 2 was comprised of 52 patients with variants in SHL-associated genes unrelated to NDD, while subgroup 3 included 29 patients with variants in genes associated with both NSHL and SHL, such as SLC26A4 and USH1C. In subgroups 2 and 3, HL was the sole initial symptom for nearly all patients (92% and 100%, respectively). Across the cohort, 99 variants in 44 genes were identified, including 36 novel variants. CONCLUSION: The frequent absence of syndromic features at presentation may lead to genetic testing or analysis restricted to NSHL-associated genes. Our findings highlight the critical role of comprehensive genomic testing in the diagnostic workup of HL, enabling earlier identification of syndromic forms and facilitating timely medical management, genetic counseling, and anticipatory care.

Dissecting biological pathways highlighted by Mendelian gene discovery has provided critical insights into the pathogenesis of Parkinson's disease (PD) and neurodegeneration. This approach ultimately catalyzes the identification of potential biomarkers and therapeutic targets. Here we identify PSMF1 as a gene implicated in parkinsonism and childhood neurodegeneration. We find that biallelic PSMF1 missense and loss-of-function variants co-segregate with phenotypes from early-onset PD to perinatal lethality with neurological manifestations across 18 pedigrees with 25 affected subjects, showing clear genotype-phenotype correlation. PSMF1 encodes the proteasome regulator PSMF1/hPI31, a highly conserved, ubiquitously expressed partner of the 20S proteasome and neurodegeneration-associated F-box-O 7 and valosin-containing proteins. We demonstrate that PSMF1 variants may affect proteasomal abundance and assembly, and are associated with alterations of mitochondrial membrane potential, respiration, dynamics and mitophagy in patient-derived fibroblasts. Furthermore, Drosophila and mouse models of PI31 loss of function exhibit age-dependent motor impairment, as well as brain-wide mitochondrial membrane depolarization and dopaminergic neurodegeneration in aged flies, and diffuse gliosis in mice. Collectively, our findings unequivocally link defective PSMF1/hPI31 to early-onset parkinsonism and neurodegeneration, and suggest proteasomal and mitochondrial dysfunction as pathogenic contributors.

Background: The alkaline ceramidase 3 (ACER3) gene encodes an enzyme that regulates bioactive lipids, including ceramides, sphingosine, and sphingosine-1-phosphate, whose balance is essential for cell viability. Biallelic ACER3 variants have been associated with early-onset progressive leukodystrophy/leukoencephalopathy, previously reported only in seven patients from five families. Methods: Exome and genome sequencing of individuals with undiagnosed neurodevelopmental disorders, combined with international data sharing, identified multiple families with biallelic ACER3 variants. Enzymatic assays, lipidomics, mutagenesis, and protein modelling were used to assess variant pathogenicity. Findings: We describe 61 patients from 56 families (54 patients from 51 newly reported families), defining the clinical and molecular spectrum of ACER3-related disease. Infantile onset occurred in 89%, with moderate (54%) or rapid (37%) progression. Premature death occurred in 28% (mean age 6.0±4.3 years); mean age of living patients was 6.2±5.0 years. Core features included developmental regression or stagnation (100%/89%), global developmental delay (72%), limb spasticity (92%), axial hypotonia (74%), dystonia (73%), feeding difficulties (72%), contractures (45%), scoliosis (39%), and epilepsy (32%). A consistent facial gestalt was observed in 9/17 assessed patients. Neuroimaging showed posterior-predominant periventricular and deep white matter abnormalities, typically with minimal cerebral atrophy (86%). Functional studies demonstrated markedly reduced or absent ceramidase activity for all tested variants, total alkaline ceramidase activity of 0–34.4% in patient fibroblasts, and accumulation of ceramides and dihydroceramides, identifying critical functional hotspots. Interpretation: Together, these findings enhance the clinical, molecular, and biochemical understanding of ACER3-related disease, underscore the critical role of ACER3 in maintaining sphingolipid homeostasis, and provide a foundation for future therapeutic avenues.

Hereditary ataxias are a heterogeneous group of neurogenetic conditions characterised by the clinical syndrome of progressive loss of coordination from neurodegeneration of the cerebellum. A commonality across the most prevalent ataxias is the underlying disease mechanism secondary to expansions of short tandem DNA repeats. There is currently an incomplete understanding of the pathogenic mechanisms of these repeat expansion disorders, a core feature of which revolves around RNA-dysregulation. In this study, we used both bulk and single nuclear RNA-sequencing to study post-mortem brain tissue of human donors with a range of repeat-expansion ataxias to reveal further mechanistic insights. We compared post-mortem paired cerebellar and frontal cortex tissue bulk RNA-sequencing data from 23 ataxia patients and 22 sex-, age-matched controls from two brain banks (spinocerebellar ataxia (SCA)1, SCA2, SCA6, SCA7, SCA17, Friedreich's ataxia (FRDA), and 7 cases with unknown molecular diagnoses). We analysed bulk RNA-sequencing data for transcript usage, differential and cell-type-specific expression to transcriptomically profile these diseases. We also generated single nuclear RNA-sequencing data of the cerebellum from donors with SCA1, SCA2, SCA6 and FRDA to decipher changes in cell type proportions in the disease state. Using this approach, we found that: (i) despite the commonalities in the genetics of ataxia, there were components of their transcriptional signatures which were distinct; (ii) there were extensive transcriptional changes evident not only in the cerebellum but also the frontal cortex in ataxia cases; (iii) activation of immune and inflammatory pathways, as well as involvement of non-neuronal cell types was a feature of all ataxias to a lesser or greater extent. This study provides a novel resource to understand the mechanisms of disease in ataxia. Furthermore, taken together, these results highlight immune pathways and the role of non-neuronal cell types as early and potentially important therapeutic targets. These findings provide a map of transcriptomic changes in ataxia to further understanding of the underlying pathogenesis. ### Competing Interest Statement The authors have declared no competing interest.

Long-read RNA sequencing has expanded our understanding of the transcriptome, revealing unannotated transcripts. However, interpreting their functional relevance remains challenging. To address this, we developed TX2P, a user-friendly tool that integrates transcriptomic and proteomic data to link RNA discoveries with protein function. Applied to epilepsy-associated genes, TX2P identified novel protein-coding transcripts, with peptide evidence supporting 17.2% transcripts and 18.0% of unique open reading frames. By seamlessly integrating transcriptomic and proteomic insights, TX2P facilitates transcriptome interpretation. ### Competing Interest Statement The authors have declared no competing interest. Aligning Science Across Parkinson's, https://ror.org/03zj4c476, ASAP-000509, ASAP-000478 BrightFocus Foundation, A2021009F

BACKGROUND:The catechol-O-methyltransferase (COMT) gene is involved in brain catecholamine metabolism, but its association with Parkinson's disease (PD) risk remains unclear. OBJECTIVE:Our aim was to investigate the relationship between COMT genetic variants and PD risk across diverse ancestries. METHODS:We analyzed COMT variants in 2251 PD patients and 2835 controls of European descent using whole-genome sequencing from the Accelerating Medicines Partnership-Parkinson Disease (AMP-PD), along with 20,427 PD patients and 11,837 controls from 10 ancestries using genotyping data from the Global Parkinson's Genetics Program (GP2). RESULTS:Using the largest case-control datasets to date, no significant enrichment of COMT risk alleles in PD patients was observed across any ancestry group after correcting for multiple testing. Among Europeans, no correlations with cognitive decline, motor function, motor complications, or time to levodopa-induced dyskinesia onset were observed. CONCLUSIONS:This study highlights the need for increased representation of diverse ancestries to better understand the role of COMT variants in PD. © 2025 The Author(s). Movement Disorders published by Wiley Periodicals LLC on behalf of International Parkinson and Movement Disorder Society.

Dissecting biological pathways highlighted by Mendelian gene discovery has provided critical insights into the pathogenesis of Parkinson's disease (PD) and neurodegeneration. This approach ultimately catalyzes the identification of potential biomarkers and therapeutic targets. Here, we identify PSMF1 as a novel gene implicated in parkinsonism and childhood neurodegeneration. We find that biallelic PSMF1 missense and loss-of-function variants co-segregate with phenotypes from early-onset PD to perinatal lethality with neurological manifestations across 17 pedigrees with 24 affected subjects, showing clear genotype-phenotype correlation. PSMF1 encodes the proteasome regulator PSMF1/PI31, a highly conserved, ubiquitously expressed partner of the 20S proteasome and neurodegeneration-associated F-box-O 7 and valosin-containing proteins. We demonstrate that PSMF1 variants impair mitochondrial membrane potential, dynamics and mitophagy, and may affect proteasomal abundance and assembly in patient-derived fibroblasts. Furthermore, Drosophila and mouse models of PSMF1 loss of function exhibit age-dependent motor impairment, as well as brain-wide mitochondrial membrane depolarization and dopaminergic neurodegeneration in aged flies, and diffuse gliosis in mice. Collectively, our findings unequivocally link defective PSMF1 to early-onset parkinsonism and neurodegeneration, and suggest proteasomal and mitochondrial dysfunction as mechanistic contributors.

OBJECTIVE:The objective was to evaluate the effects of deep brain stimulation (DBS) in an international cohort of patients with VPS16-related dystonia. METHODS:This observational study collected preoperative and postoperative demographic, clinical, stimulation, genetic, neuroimaging, and neurophysiological data of medically refractory DYT-VPS16 patients with implanted DBS in 10 international centers. Motor symptoms and disability outcomes were assessed using the Burke-Fahn-Marsden Dystonia Rating Scale Motor (BFMDRS-M) and Disability (BFMDRS-D) scales. A cut-off threshold for considering response to DBS was set at 25% of BFMDRS-M improvement at the last follow-up (FU) compared to baseline. RESULTS:The cohort consisted of 26 participants (17 men, 65.4%). Age at dystonia onset and surgery was 17.8 ± 10.9 and 35.3 ± 14.8 years, respectively. At the last FU, 102.5 ± 57.3 months (range, 2-216), the mean BFMDRS-M improvement was 41.6 ± 37.3% (26/26 patients) and 34.8 ± 42.6% for the BFMDRS-D (23/26 patients). Most patients (19/26, 73%) were considered responders. Higher motor improvement was associated with stimulation of the ventroposterior portion of the internal globus pallidus. A significant inverse relationship was observed between improvement in BFMDRS-M at last FU, and the presence of spasticity (p = 0.027) and fixed skeletal deformities (p = 0.001) before surgery. Non-responders had a younger age at disease onset and at implantation, shorter disease duration at DBS surgery, and higher baseline BFMDRS scores. INTERPRETATION:DBS was an effective treatment for three-quarters of patients with pathogenic VPS16 variants in our cohort. Mean motor improvement was most pronounced at the 1-year FU, but persisted at the last FU despite disease progression. ANN NEUROL 2025;98:711-725.

MDGA2 encodes a membrane-associated protein that is critical for regulating glutamatergic synapse development, modulating neuroligins (Nlgns), and maintaining the balance between excitatory and inhibitory synapses. Although MDGA2 has been extensively studied in murine and cellular models, its association with human developmental disorders has not been established to date. Through exome sequencing we identified six distinct homozygous loss-of-function MDGA2 variants in eight individuals from six consanguineous families, all presenting with developmental and epileptic encephalopathy (DEE). Clinically, these patients exhibited infantile hypotonia, severe neurodevelopmental delay, intractable seizures, progressive brain atrophy, and consistent dysmorphic features, including high anterior hairline, high-arched eyebrows, broad nasal ridge, tented upper lip, and large, low-set ears. Functional studies of three representative nonsense variants in mammalian expression systems and hippocampal cultured neurons revealed impaired MDGA2 membrane trafficking and disrupted Nlgn1 interaction, leading to defective excitatory synapse formation, synaptic transmission, and synaptic strength. Altogether, our findings definitively establish MDGA2 as a novel gene for autosomal recessive DEE subtype, with the pathogenesis explained by loss-of-function mechanism. This discovery underscores the previously unrecognized role of MDGA2 in human synaptic development and regulation, significantly expanding our understanding of the genetic architecture of DEE. ### Competing Interest Statement VAY is founder, shareholder and managing director of OmicsDiscoveries GmbH. ### Funding Statement H.M. was supported by the Wellcome Trust grant 220906/Z/20/Z and UCL Global Engagement Fund scheme 2023. H.K. was supported by the National Research Foundation of Korea (NRF) funded by the Ministry of Science and Future Planning (RS-2024-00339642). J.W.U. was supported by the NRF funded by the Ministry of Science and Future Planning (RS-2023-NR076948). J.K. was supported by the NRF funded by the Ministry of Science and Future Planning (RS-2022-NR070708). M.Z. was funded by GERF-STDF: 33650, STDF, Egypt. H.H. was funded by the Wellcome Trust, MRC, MSA Trust, National Institute for Health Research University College London Hospitals Biomedical Research Centre (NIHR-BRC), Michael J Fox Foundation (MJFF), Fidelity Trust, Rosetrees Trust, Dolby Family Fund, Alzheimer's Research UK (ARUK), MSA Coalition, Parkinson's Disease Society, Parkinson's Foundation, Guarantors of Brain, Cerebral Palsy Alliance, FARA, EAN, Victoria Brain Bank, NIH NeuroBioBank, Queen Square BrainBank, and MRC Brainbank Network. V.A.Y. and J.G. were funded by the Deutsche Forschungsgemeinschaft (DFG, German Research Foundation) via the project NFDI 1/1 "GHGA - German Human Genome-Phenome Archive" (#441914366). ERDERA has received funding from the European Union Horizon Europe research and innovation programme under grant agreement No. 101156595. The TUM IT infrastructure was co-funded by the Deutsche Forschungsgemeinschaft (DFG, German Research Foundation) , Project-ID 461264291 ### Author Declarations I confirm all relevant ethical guidelines have been followed, and any necessary IRB and/or ethics committee approvals have been obtained. Yes The details of the IRB/oversight body that provided approval or exemption for the research described are given below: Research Ethics Committee, Institute of Neurology, University College London (ION UCL) (07/Q0512/26) I confirm that all necessary patient/participant consent has been obtained and the appropriate institutional forms have been archived, and that any patient/participant/sample identifiers included were not known to anyone (e.g., hospital staff, patients or participants themselves) outside the research group so cannot be used to identify individuals. Yes I understand that all clinical trials and any other prospective interventional studies must be registered with an ICMJE-approved registry, such as ClinicalTrials.gov. I confirm that any such study reported in the manuscript has been registered and the trial registration ID is provided (note: if posting a prospective study registered retrospectively, please provide a statement in the trial ID field explaining why the study was not registered in advance). Yes I have followed all appropriate research reporting guidelines, such as any relevant EQUATOR Network research reporting checklist(s) and other pertinent material, if applicable. Yes The data that support the findings of this study are available within the paper and in the supplemental information. Whole exome sequencing data are not publicly available due to privacy or ethical restrictions.

FITM2 encodes fat-storage inducing transmembrane protein 2 (FIT2), a lipid diphosphatase in the ER that cleaves acyl-CoAs and is crucial for ER homeostasis. In humans, homozygous null mutations in FITM2 are associated with a syndrome characterized by deafness and dystonia. Here, we report two families with hereditary spastic paraplegia (HSP) in whom exome sequencing revealed compound heterozygosity for FITM2 mutations. In each family, the affected probands carry one putative null allele and one G100R missense allele. Functional analyses demonstrated that the G100R allele is hypomorphic, with FIT2 protein levels reduced to 20% of wild type, leading to proportionately decreased enzyme activity. The occurrence of similar HSP disease phenotypes and the same hypomorphic mutation in these families suggests that the G100R mutation and its associated reduced enzyme activity represent a newly recognized clinical manifestation of FITM2 mutations, expanding the spectrum of conditions associated with this gene.

Partial phenotypic overlap has been suggested between multiple system atrophy and spinocerebellar ataxia 27B, the autosomal dominant ataxia caused by an intronic GAA•TTC repeat expansion in FGF14. In this study, we investigated the frequency of FGF14 GAA•TTC repeat expansion in clinically diagnosed and pathologically confirmed multiple system atrophy cases. We screened 657 multiple system atrophy cases (193 clinically diagnosed and 464 pathologically confirmed) and 1003 controls. The FGF14 repeat locus was genotyped using long-range PCR and bidirectional repeat-primed PCRs, and expansions were confirmed with targeted long-read Oxford Nanopore Technologies sequencing. We identified 19 multiple system atrophy cases carrying an FGF14 GAA≥250 expansion (2.89%, n = 19/657), a significantly higher frequency than in controls (1.40%, n = 12/1003) (P = 0.04). Long-read Oxford Nanopore Technologies sequencing confirmed repeat sizes and polymorphisms detected by PCR, with high concordance (Pearson's r = 0.99, P < 0.0001). Seven multiple system atrophy patients had a pathogenic FGF14 GAA≥300 expansion (five pathologically confirmed and two clinically diagnosed), and 12 had intermediate GAA250-299 expansion (six pathologically confirmed and six clinically diagnosed). A similar proportion of cerebellar-predominant and parkinsonism-predominant multiple system atrophy cases had FGF14 expansions. Multiple system atrophy patients carrying an FGF14 GAA≥250 expansion exhibited severe gait ataxia, autonomic dysfunction and parkinsonism, in keeping with a multiple system atrophy phenotype, with a faster progression to falls (P = 0.03) and regular wheelchair use (P = 0.02) in comparison to the multiple system atrophy cases without FGF14 GAA expansion. The length of the GAA•TTC repeat expansion lengths was inversely correlated with survival in multiple system atrophy patients (r = -0.67; P = 0.02) but not with age of onset. Therefore, screening for FGF14 GAA•TTC repeat expansion should be considered for multiple system atrophy patients with rapid loss of mobility and for complete diagnostic accuracy at inclusion in disease-modifying multiple system atrophy drug trials.

SCN3B encodes the beta 3 auxiliary subunit, essential for voltage-gated Na+ (Nav) channel trafficking and gating. Although SCN3B has been associated with cardiac disorders, a link with neurodevelopmental disorders (NDD) has not been established. Using a genotype-first approach, we identified homozygous truncating variants (c.281G>A-beta 3(W94*), c.584 + 1G>A-beta 3(S196*)) in 2 consanguineous Pakistani families, leading to global developmental delay, intellectual disability and autism, with severe cognitive impairment, ataxia, and seizures in the case of beta 3(W94*). Electrophysiological analysis revealed subtype-specific gating alterations on multiple brain Nav channel subtypes. This is the first report linking SCN3B mutations to NDD, expanding our understanding of Nav channelopathies.

BACKGROUND:Leucine-rich repeat kinase 2 (LRRK2) p.L1795F variant was proposed as a genetic risk factor for Parkinson's disease (PD). However, its prevalence, phenotype, and origin remain unknown. OBJECTIVE:The aim was to evaluate the frequency and phenotype of p.L1795F in early-onset PD (EOPD) and familial PD compared to healthy controls (HC) in Central Europe. METHODS:Whole-exome sequencing was used to screen 219 EOPD and familial PD patients of Central Europeans compared to HC. Sanger sequencing assessed segregation. Detailed clinical phenotype was evaluated for all positive carriers. RESULTS:p.L1795F was identified in 1.37% (3/219) and 3.23% of familial cases (3/93), with no carriers among HCs (0/303). Segregation analysis confirmed association with PD. Carriers were traced to the eastern Slovak-Hungarian region. It also appears to be associated with a more aggressive phenotype. CONCLUSION:Our data indicate that p.L1795F contributes to PD in Central Europe. Further exploration in larger cohorts is warranted to establish its contribution to global PD risk.

Background/Objectives: Short tandem repeat expansions are the most common cause of inherited neurological diseases. These disorders are clinically and genetically heterogeneous, such as in myotonic dystrophy and spinocerebellar ataxia, and they are caused by different repeat motifs in different genomic locations. Major advances in bioinformatic tools used to detect repeat expansions from short read sequencing data in the last few years have led to the implementation of these workflows into next generation sequencing pipelines in healthcare. Here, we aimed to evaluate the clinical utility of analysing repeat expansions through exome sequencing in a large cohort of genetically undiagnosed patients with neurological disorders. Methods: We here analyse 27 disease-causing DNA repeats found in the coding, intronic and untranslated regions in 12,496 exomes in patients with a range of neurogenetic conditions. Results: We identified—and validated by polymerase chain reaction—29 repeat expansions across a range of loci, 48% (n = 14) of which were diagnostic. We then analysed the genotyping performance across all repeat loci and found that, despite high coverage in most repeats in coding regions, some loci had low genotyping rates, such as those that cause spinocerebellar ataxia 2 (ATXN2, 0.1–8.4%) and Huntington disease (HTT, 0.2–58.2%), depending on the capture kit. Conversely, while most intronic repeats were not genotyped, we found a high genotyping rate in the intronic locus that causes spinocerebellar ataxia 36 (NOP56, 30.1–98.3%) and in the one that causes myotonic dystrophy type 1 (DMPK, myotonic dystrophy type 1). Conclusions: We show that the key factors that influence the genotyping rate of repeat expansion loci analysis are the sequencing read length and exome capture kit. These results provide important information about the performance of exome sequencing as a genetic test for repeat expansion disorders.

Developmental Delay with Gastrointestinal, Cardiovascular, Genitourinary, and Skeletal Abnormalities syndrome (DEGCAGS, MIM #619488) is caused by biallelic, loss-of-function (LoF) ZNF699 variants, and is characterized by variable neurodevelopmental disability, discordant organ anomalies among full siblings and infant mortality. ZNF699 encodes a KRAB zinc finger protein of unknown function. We aimed to investigate the genotype-phenotype spectrum of DEGCAGS and the possibility of a diagnostic DNA methylation episignature, to facilitate the diagnosis of a highly variable condition lacking pathognomonic clinical findings. We collected data on 30 affected individuals (12 new). GestaltMatcher analyzed fifty-three facial photographs from five individuals. In nine individuals, methylation profiling of blood-DNA was performed, and a classification model was constructed to differentiate DEGCAGS from controls. We expand the ZNF699-related molecular spectrum and show that biallelic, LoF, ZNF699 variants cause unique clinical findings with age-related presentation and a similar facial gestalt. We also identified a robust episignature for DEGCAGS syndrome. DEGCAGS syndrome is a clinically variable recessive syndrome even among siblings with a distinct methylation episignature which can be used as a screening, diagnostic and classification tool for ZNF699 variants. Analysis of differentially methylated regions suggested an effect on genes potentially implicated in the syndrome’s pathogenesis.

Primary familial brain calcification (PFBC) is characterized by calcium deposition in the brain, causing progressive movement disorders, psychiatric symptoms, and cognitive decline. PFBC is a heterogeneous disorder currently linked to variants in six different genes, but most patients remain genetically undiagnosed. Here, we identify biallelic NAA60 variants in ten individuals from seven families with autosomal recessive PFBC. The NAA60 variants lead to loss-of-function with lack of protein N-terminal (Nt)-acetylation activity. We show that the phosphate importer SLC20A2 is a substrate of NAA60 in vitro. In cells, loss of NAA60 caused reduced surface levels of SLC20A2 and a reduction in extracellular phosphate uptake. This study establishes NAA60 as a causal gene for PFBC, provides a possible biochemical explanation of its disease-causing mechanisms and underscores NAA60-mediated Nt-acetylation of transmembrane proteins as a fundamental process for healthy neurobiological functioning.

Retinoblastoma (RB) proteins are highly conserved transcriptional regulators that play important roles during development by regulating cell-cycle gene expression. RBL2 dysfunction has been linked to a severe neurodevelopmental disorder. However, to date, clinical features have only been described in six individuals carrying five biallelic predicted loss of function (pLOF) variants. To define the phenotypic effects of RBL2 mutations in detail, we identified and clinically characterized a cohort of 28 patients from 18 families carrying LOF variants in RBL2 , including fourteen new variants that substantially broaden the molecular spectrum. The clinical presentation of affected individuals is characterized by a range of neurological and developmental abnormalities. Global developmental delay and intellectual disability were uniformly observed, ranging from moderate to profound and involving lack of acquisition of key motor and speech milestones in most patients. Frequent features included postnatal microcephaly, infantile hypotonia, aggressive behaviour, stereotypic movements and non-specific dysmorphic features. Common neuroimaging features were cerebral atrophy, white matter volume loss, corpus callosum hypoplasia and cerebellar atrophy. In parallel, we used the fruit fly, Drosophila melanogaster , to investigate how disruption of the conserved RBL2 orthologueue Rbf impacts nervous system function and development. We found that Drosophila Rbf LOF mutants recapitulate several features of patients harboring RBL2 variants, including alterations in the head and brain morphology reminiscent of microcephaly, and perturbed locomotor behaviour. Surprisingly, in addition to its known role in controlling tissue growth during development, we find that continued Rbf expression is also required in fully differentiated post-mitotic neurons for normal locomotion in Drosophila , and that adult-stage neuronal re-expression of Rbf is sufficient to rescue Rbf mutant locomotor defects. Taken together, this study provides a clinical and experimental basis to understand genotype-phenotype correlations in an RBL2 -linked neurodevelopmental disorder and suggests that restoring RBL2 expression through gene therapy approaches may ameliorate aspects of RBL2 LOF patient symptoms.

Summary The role of the SNCA gene locus in driving Parkinson’s disease (PD) through rare and common genetic variation is well-recognized, but the transcriptional diversity of SNCA in vulnerable cell types remains unclear. We performed SNCA long-read RNA sequencing in human dopaminergic neurons and show that annotated SNCA transcripts account for only 5% of expression. Rather, the majority of expression (75%) at the SNCA locus originates from transcripts with alternative 5’ and 3’ untranslated regions. Importantly, 10% originates from transcripts encoding open reading frames not previously annotated, which are translated and detectable in human postmortem brain. Defining the 3’ untranslated regions enabled the rational design of antisense oligonucleotides targeting the majority of SNCA transcripts, leading to the effective reversal of PD pathology, including protein aggregation, mitochondrial dysfunction, and toxicity. Resolving the complexity of the SNCA transcriptional landscape impacts RNA therapies and highlights differences in protein isoforms and their contribution to disease.

Malaria is a burdensome disease to humanity caused chiefly by the still poorly understood parasite genus Plasmodium . Much of the pathogenic success of these and other related parasites is due to the presence of the apicoplast, a comparatively poorly characterised biosynthetic organelle containing many proteins of unknown function. Here we present AlphaFold2 protein structure predictions together with further in silico analyses to infer molecular functions for the three uncharacterised transmembrane apicoplast proteins PF3D7\_0622700, PF3D7\_0908100 and PF3D7\_1021300. The targets PF3D7\_0622700 and PF3D7\_0908100 are shown herein to belong to the polytopic Major Facilitator and Cation-Proton Antiporter and Anion Transporter superfamilies respectively, confirming previous suspicions for PF3D7\_0908100 of a transporter function. Importantly, our docking screens further suggest pyridoxal-5-phosphate may be transported by PF3D7\_0622700, and PF3D7\_0908100 likely transports a larger negatively charged metabolite. These findings will help direct experimental assays to confirm what apicoplast metabolites these proteins may transport. PF3D7_1021300 is proposed to possess a six transmembrane alpha-helix domain of a currently unknown fold which may also possess a transporter molecular function. This work highlights the power of high accuracy protein structure predictions to illuminate proteins of unknown structure and function. ### Competing Interest Statement The authors have declared no competing interest.