Genotype-phenotype associations in 1018 individuals with SCN1A-related epilepsies.

OBJECTIVE: SCN1A variants are associated with epilepsy syndromes ranging from mild genetic epilepsy with febrile seizures plus (GEFS+) to severe Dravet syndrome (DS). Many variants are de novo, making early phenotype prediction difficult, and genotype-phenotype associations remain poorly understood. METHODS: We assessed data from a retrospective cohort of 1018 individuals with SCN1A-related epilepsies. We explored relationships between variant characteristics (position, in silico prediction scores: Combined Annotation Dependent Depletion (CADD), Rare Exome Variant Ensemble Learner (REVEL), SCN1A genetic score), seizure characteristics, and epilepsy phenotype. RESULTS: DS had earlier seizure onset than other GEFS+ phenotypes (5.3 vs. 12.0 months, p < .001). In silico variant scores were higher in DS versus GEFS+ (p < .001). Patients with missense variants in functionally important regions (conserved N-terminus, S4-S6) exhibited earlier seizure onset (6.0 vs. 7.0 months, p = .003) and were more likely to have DS (280/340); those with missense variants in nonconserved regions had later onset (10.0 vs. 7.0 months, p = .036) and were more likely to have GEFS+ (15/29, χ2 = 19.16, p < .001). A minority of protein-truncating variants were associated with GEFS+ (10/393) and more likely to be located in the proximal first and last exon coding regions than elsewhere in the gene (9.7% vs. 1.0%, p < .001). Carriers of the same missense variant exhibited less variability in age at seizure onset compared with carriers of different missense variants for both DS (1.9 vs. 2.9 months, p = .001) and GEFS+ (8.0 vs. 11.0 months, p = .043). Status epilepticus as presenting seizure type is a highly specific (95.2%) but nonsensitive (32.7%) feature of DS. SIGNIFICANCE: Understanding genotype-phenotype associations in SCN1A-related epilepsies is critical for early diagnosis and management. We demonstrate an earlier disease onset in patients with missense variants in important functional regions, the occurrence of GEFS+ truncating variants, and the value of in silico prediction scores. Status epilepticus as initial seizure type is a highly specific, but not sensitive, early feature of DS.

Expanding the phenotypic spectrum of CLCN2-related leucoencephalopathy and ataxia.

Mutations in CLCN2 are a rare cause of autosomal recessive leucoencephalopathy with ataxia and specific imaging abnormalities. Very few cases have been reported to date. Here, we describe the clinical and imaging phenotype of 12 additional CLCN2 patients and expand the known phenotypic spectrum of this disorder. Informed consent was obtained for all patients. Patients underwent either whole-exome sequencing or focused/panel-based sequencing to identify variants. Twelve patients with biallelic CLCN2 variants are described. This includes three novel likely pathogenic missense variants. All patients demonstrated typical MRI changes, including hyperintensity on T2-weighted images in the posterior limbs of the internal capsules, midbrain cerebral peduncles, middle cerebellar peduncles and cerebral white matter. Clinical features included a variable combination of ataxia, headache, spasticity, seizures and other symptoms with a broad range of age of onset. This report is now the largest case series of patients with CLCN2-related leucoencephalopathy and reinforces the finding that, although the imaging appearance is uniform, the phenotypic expression of this disorder is highly heterogeneous. Our findings expand the phenotypic spectrum of CLCN2-related leucoencephalopathy by adding prominent seizures, severe spastic paraplegia and developmental delay.

Intronic FGF14 GAA repeat expansions are a common cause of ataxia syndromes with neuropathy and bilateral vestibulopathy.

BACKGROUND: Intronic GAA repeat expansions in the fibroblast growth factor 14 gene (FGF14) have recently been identified as a common cause of ataxia with potential phenotypic overlap with RFC1-related cerebellar ataxia, neuropathy and vestibular areflexia syndrome (CANVAS). Our objective was to report on the frequency of intronic FGF14 GAA repeat expansions in patients with an unexplained CANVAS-like phenotype. METHODS: We recruited 45 patients negative for biallelic RFC1 repeat expansions with a combination of cerebellar ataxia plus peripheral neuropathy and/or bilateral vestibulopathy (BVP), and genotyped the FGF14 repeat locus. Phenotypic features of GAA-FGF14-positive versus GAA-FGF14-negative patients were compared. RESULTS: Frequency of FGF14 GAA repeat expansions was 38% (17/45) in the entire cohort, 38% (5/13) in the subgroup with cerebellar ataxia plus polyneuropathy, 43% (9/21) in the subgroup with cerebellar ataxia plus BVP and 27% (3/11) in patients with all three features. BVP was observed in 75% (12/16) of GAA-FGF14-positive patients. Polyneuropathy was at most mild and of mixed sensorimotor type in six of eight GAA-FGF14-positive patients. Family history of ataxia (59% vs 15%; p=0.007) was significantly more frequent and permanent cerebellar dysarthria (12% vs 54%; p=0.009) significantly less frequent in GAA-FGF14-positive than in GAA-FGF14-negative patients. Age at onset was inversely correlated to the size of the repeat expansion (Pearson's r, -0.67; R2=0.45; p=0.0031). CONCLUSIONS: GAA-FGF14-related disease is a common cause of cerebellar ataxia with polyneuropathy and/or BVP, and should be included in the differential diagnosis of RFC1 CANVAS and disease spectrum.

Further characterisation of ARX-related disorders in females due to inherited or de novo variants.

The Aristaless-related homeobox (ARX) gene is located on the X chromosome and encodes a transcription factor that is essential for brain development. While the clinical spectrum of ARX-related disorders is well described in males, from X linked lissencephaly with abnormal genitalia syndrome to syndromic and non-syndromic intellectual disability (ID), its phenotypic delineation in females is incomplete. Carrier females in ARX families are usually asymptomatic, but ID has been reported in some of them, as well as in others with de novo variants. In this study, we collected the clinical and molecular data of 10 unpublished female patients with de novo ARX pathogenic variants and reviewed the data of 63 females from the literature with either de novo variants (n=10), inherited variants (n=33) or variants of unknown inheritance (n=20). Altogether, the clinical spectrum of females with heterozygous pathogenic ARX variants is broad: 42.5% are asymptomatic, 16.4% have isolated agenesis of the corpus callosum (ACC) or mild symptoms (learning disabilities, autism spectrum disorder, drug-responsive epilepsy) without ID, whereas 41% present with a severe phenotype (ie, ID or developmental and epileptic encephalopathy (DEE)). The ID/DEE phenotype was significantly more prevalent in females carrying de novo variants (75%, n=15/20) versus in those carrying inherited variants (27.3%, n=9/33). ACC was observed in 66.7% (n=24/36) of females who underwent a brain MRI. By refining the clinical spectrum of females carrying ARX pathogenic variants, we show that ID is a frequent sign in females with this X linked condition.

Episignatures in practice: independent evaluation of published episignatures for the molecular diagnostics of ten neurodevelopmental disorders.

Variants of uncertain significance (VUS) are a significant issue for the molecular diagnosis of rare diseases. The publication of episignatures as effective biomarkers of certain Mendelian neurodevelopmental disorders has raised hopes to help classify VUS. However, prediction abilities of most published episignatures have not been independently investigated yet, which is a prerequisite for an informed and rigorous use in a diagnostic setting. We generated DNA methylation data from 101 carriers of (likely) pathogenic variants in ten different genes, 57 VUS carriers, and 25 healthy controls. Combining published episignature information and new validation data with a k-nearest-neighbour classifier within a leave-one-out scheme, we provide unbiased specificity and sensitivity estimates for each of the signatures. Our procedure reached 100% specificity, but the sensitivities unexpectedly spanned a very large spectrum. While ATRX, DNMT3A, KMT2D, and NSD1 signatures displayed a 100% sensitivity, CREBBP-RSTS and one of the CHD8 signatures reached <40% sensitivity on our dataset. Remaining Cornelia de Lange syndrome, KMT2A, KDM5C and CHD7 signatures reached 70-100% sensitivity at best with unstable performances, suffering from heterogeneous methylation profiles among cases and rare discordant samples. Our results call for cautiousness and demonstrate that episignatures do not perform equally well. Some signatures are ready for confident use in a diagnostic setting. Yet, it is imperative to characterise the actual validity perimeter and interpretation of each episignature with the help of larger validation sample sizes and in a broader set of episignatures.

Loss-of-function variants in ZEB1 cause dominant anomalies of the corpus callosum with favourable cognitive prognosis.

BACKGROUND: The neurodevelopmental prognosis of anomalies of the corpus callosum (ACC), one of the most frequent brain malformations, varies extremely, ranging from normal development to profound intellectual disability (ID). Numerous genes are known to cause syndromic ACC with ID, whereas the genetics of ACC without ID remains poorly deciphered. METHODS: Through a collaborative work, we describe here ZEB1, a gene previously involved in an ophthalmological condition called type 3 posterior polymorphous corneal dystrophy, as a new dominant gene of ACC. We report a series of nine individuals with ACC (including three fetuses terminated due to ACC) carrying a ZEB1 heterozygous loss-of-function (LoF) variant, identified by exome sequencing. RESULTS: In five cases, the variant was inherited from a parent with a normal corpus callosum, which illustrates the incomplete penetrance of ACC in individuals with an LoF in ZEB1. All patients reported normal schooling and none of them had ID. Neuropsychological assessment in six patients showed either normal functioning or heterogeneous cognition. Moreover, two patients had a bicornuate uterus, three had a cardiovascular anomaly and four had macrocephaly at birth, which suggests a larger spectrum of malformations related to ZEB1. CONCLUSION: This study shows ZEB1 LoF variants cause dominantly inherited ACC without ID and extends the extraocular phenotype related to this gene.

Loss of phospholipase PLAAT3 causes a mixed lipodystrophic and neurological syndrome due to impaired PPARγ signaling.

Phospholipase A/acyltransferase 3 (PLAAT3) is a phospholipid-modifying enzyme predominantly expressed in neural and white adipose tissue (WAT). It is a potential drug target for metabolic syndrome, as Plaat3 deficiency in mice protects against diet-induced obesity. We identified seven patients from four unrelated consanguineous families, with homozygous loss-of-function variants in PLAAT3, who presented with a lipodystrophy syndrome with loss of fat varying from partial to generalized and associated with metabolic complications, as well as variable neurological features including demyelinating neuropathy and intellectual disability. Multi-omics analysis of mouse Plaat3-/- and patient-derived WAT showed enrichment of arachidonic acid-containing membrane phospholipids and a strong decrease in the signaling of peroxisome proliferator-activated receptor gamma (PPARγ), the master regulator of adipocyte differentiation. Accordingly, CRISPR-Cas9-mediated PLAAT3 inactivation in human adipose stem cells induced insulin resistance, altered adipocyte differentiation with decreased lipid droplet formation and reduced the expression of adipogenic and mature adipocyte markers, including PPARγ. These findings establish PLAAT3 deficiency as a hereditary lipodystrophy syndrome with neurological manifestations, caused by a PPARγ-dependent defect in WAT differentiation and function.

Effects of paternal and chronological age on BEGAIN methylation and its possible role in autism.

Children from old fathers carry an increased risk for autism spectrum (ASD) and other neurodevelopmental disorders, which may at least partially be mediated by paternal age effects on the sperm epigenome. The brain enriched guanylate kinase associated (BEGAIN) protein is involved in protein-protein interactions at and transmission across synapses. Since several epigenome-wide methylation screens reported a paternal age effect on sperm BEGAIN methylation, here we confirmed a significant negative correlation between BEGAIN promoter methylation and paternal age, using more sensitive bisulfite pyrosequencing and a larger number of sperm samples. Paternal age-associated BEGAIN hypomethylation was also observed in fetal cord blood (FCB) of male but not of female offspring. There was no comparable maternal age effect on FCB methylation. In addition, we found a significant negative correlation between BEGAIN methylation and chronological age (ranging from 1 to 70 years) in peripheral blood samples of male but not of female donors. BEGAIN hypomethylation was more pronounced in male children, adolescents and adults suffering from ASD compared to controls. Both genetic variation (CC genotype of SNP rs7141087) and epigenetic factors may contribute to BEGAIN promoter hypomethylation. The age- and sex-specific BEGAIN methylation trajectories in the male germ line and somatic tissues, in particular the brain, support a role of this gene in ASD development.

Correction: Association of Rare Genetic Variants in Opioid Receptors with Tourette Syndrome.

[This corrects the article DOI: 10.5334/tohm.464.].

Bi-allelic variants of FILIP1 cause congenital myopathy, dysmorphism and neurological defects.

Filamin-A-interacting protein 1 (FILIP1) is a structural protein that is involved in neuronal and muscle function and integrity and interacts with FLNa and FLNc. Pathogenic variants in filamin-encoding genes have been linked to neurological disorders (FLNA) and muscle diseases characterized by myofibrillar perturbations (FLNC), but human diseases associated with FILIP1 variants have not yet been described. Here, we report on five patients from four unrelated consanguineous families with homozygous FILIP1 variants (two nonsense and two missense). Functional studies indicated altered stability of the FILIP1 protein carrying the p.[Pro1133Leu] variant. Patients exhibit a broad spectrum of neurological symptoms including brain malformations, neurodevelopmental delay, muscle weakness and pathology and dysmorphic features. Electron and immunofluorescence microscopy on the muscle biopsy derived from the patient harbouring the homozygous p.[Pro1133Leu] missense variant revealed core-like zones of myofibrillar disintegration, autophagic vacuoles and accumulation of FLNc. Proteomic studies on the fibroblasts derived from the same patient showed dysregulation of a variety of proteins including FLNc and alpha-B-crystallin, a finding (confirmed by immunofluorescence) which is in line with the manifestation of symptoms associated with the syndromic phenotype of FILIP1opathy. The combined findings of this study show that the loss of functional FILIP1 leads to a recessive disorder characterized by neurological and muscular manifestations as well as dysmorphic features accompanied by perturbed proteostasis and myopathology.

Genetics of familial adult myoclonus epilepsy: From linkage studies to noncoding repeat expansions.

Familial adult myoclonus epilepsy (FAME) is a genetic epilepsy syndrome that for many years has resisted understanding of its underlying molecular cause. This review covers the history of FAME genetic studies worldwide, starting with linkage and culminating in the discovery of noncoding TTTTA and inserted TTTCA pentanucleotide repeat expansions within six different genes to date (SAMD12, STARD7, MARCHF6, YEATS2, TNRC6A, and RAPGEF2). FAME occurs worldwide; however, repeat expansions in particular genes have regional geographical distributions. FAME repeat expansions are dynamic in nature, changing in length and structure within germline and somatic tissues. This variation poses challenges for molecular diagnosis such that molecular methods used to identify FAME repeat expansions typically require a trade-off between cost and efficiency. A rigorous evaluation of the sensitivity and specificity of each molecular approach remains to be performed. The origin of FAME repeat expansions and the genetic and environmental factors that modulate repeat variability are not well defined. Longer repeats and particular arrangements of the TTTTA and TTTCA motifs within an expansion are correlated with earlier onset and increased severity of disease. Other factors such as maternal or paternal inheritance, parental age, and repeat length alone have been suggested to influence repeat variation; however, further research is required to confirm this. The history of FAME genetics to the present is a chronicle of perseverance and predominantly collaborative efforts that yielded a successful outcome. The discovery of FAME repeats will spark progress toward a deeper understanding of the molecular pathogenesis of FAME, discovery of new loci, and development of cell and animal models.

Individual perception of environmental factors that influence lower limbs spasticity in inherited spastic paraparesis.

BACKGROUND: Phenotypic variability is a consistent finding in neurogenetics and therefore applicable to hereditary spastic paraparesis. Identifying reasons for this variability is a challenge. We hypothesized that, in addition to genetic modifiers, extrinsic factors influence variability. OBJECTIVES: Our aim was to describe the clinical variability in hereditary spastic paraparesis from the person's perspective. Our goals were to identify individual and environmental factors that influence muscle tone disorders and derive interventions which could improve spasticity. METHODS: This study was based on self-assessments with questions on nominal and ordinal scales completed by participants with hereditary spastic paraparesis. A questionnaire was completed either in-person in the clinic or electronically via lay organization websites. RESULTS: Among the 325 responders, most had SPG4/SPAST (n = 182, 56%) with a mean age at onset of 31.7 (SD 16.7) years and a mean disease duration of 23 (SD 13.6) years at the time of participation. The 2 factors identified as improving spasticity for > 50% of the responders were physiotherapy (193/325, 59%), and superficial warming (172/308, 55%). Half of the responders (n = 164, 50%) performed physical activity at least once a month and up to once a week. Participants who reported physiotherapy as effective were significantly more satisfied with ≥ 3 sessions per week. Psychologically stressful situations (246/319, 77%) and cold temperatures (202/319, 63%) exacerbated spasticity for most participants. CONCLUSION: Participants perceived that physiotherapy reduced spasticity and that the impact of physiotherapy on spasticity was much greater than other medical interventions. Therefore, people should be encouraged to practice physical activity at least 3 times per week. This study reported participants' opinions: in hereditary spastic paraparesis only functional treatments exist, therefore the participant's expertise is of particular importance.

De novo variants in CNOT9 cause a neurodevelopmental disorder with or without epilepsy.

PURPOSE: The study aimed to clinically and molecularly characterize the neurodevelopmental disorder associated with heterozygous de novo variants in CNOT9. METHODS: Individuals were clinically examined. Variants were identified using exome or genome sequencing. These variants were evaluated using in silico predictions, and their functional relevance was further assessed by molecular models and research in the literature. The variants have been classified according to the criteria of the American College of Medical Genetics. RESULTS: We report on 7 individuals carrying de novo missense variants in CNOT9, p.(Arg46Gly), p.(Pro131Leu), and p.(Arg227His), and, recurrent in 4 unrelated individuals, p.(Arg292Trp). All affected persons have developmental delay/intellectual disability, with 5 of them showing seizures. Other symptoms include muscular hypotonia, facial dysmorphism, and behavioral abnormalities. Molecular modeling predicted that the variants are damaging and would lead to reduced protein stability or impaired recognition of interaction partners. Functional analyses in previous studies showed a pathogenic effect of p.(Pro131Leu) and p.(Arg227His). CONCLUSION: We propose CNOT9 as a novel gene for neurodevelopmental disorder and epilepsy.

Genome-wide Association Study Points to Novel Locus for Gilles de la Tourette Syndrome.

BACKGROUND: Tourette syndrome (TS) is a childhood-onset neurodevelopmental disorder of complex genetic architecture and is characterized by multiple motor tics and at least one vocal tic persisting for more than 1 year. METHODS: We performed a genome-wide meta-analysis integrating a novel TS cohort with previously published data, resulting in a sample size of 6133 individuals with TS and 13,565 ancestry-matched control participants. RESULTS: We identified a genome-wide significant locus on chromosome 5q15. Integration of expression quantitative trait locus, Hi-C (high-throughput chromosome conformation capture), and genome-wide association study data implicated the NR2F1 gene and associated long noncoding RNAs within the 5q15 locus. Heritability partitioning identified statistically significant enrichment in brain tissue histone marks, while polygenic risk scoring of brain volume data identified statistically significant associations with right and left thalamus volumes and right putamen volume. CONCLUSIONS: Our work presents novel insights into the neurobiology of TS, thereby opening up new directions for future studies.

Novel Variants of SOX4 in Patients with Intellectual Disability.

SOX4 is a transcription factor with pleiotropic functions required for different developmental processes, such as corticogenesis. As with all SOX proteins, it contains a conserved high mobility group (HMG) and exerts its function via interaction with other transcription factors, such as POU3F2. Recently, pathogenic SOX4 variants have been identified in several patients who had clinical features overlapping with Coffin-Siris syndrome. In this study, we identified three novel variants in unrelated patients with intellectual disability, two of which were de novo (c.79G>T, p.Glu27*; c.182G>A p.Arg61Gln) and one inherited (c.355C>T, p.His119Tyr). All three variants affected the HMG box and were suspected to influence SOX4 function. We investigated the effects of these variants on transcriptional activation by co-expressing either wildtype (wt) or mutant SOX4 with its co-activator POU3F2 and measuring their activity in reporter assays. All variants abolished SOX4 activity. While our experiments provide further support for the pathogenicity of SOX4 loss-of-function (LOF) variants as a cause of syndromic intellectual disability (ID), our results also indicate incomplete penetrance associated with one variant. These findings will improve classification of novel, putatively pathogenic SOX4 variants.

A novel FAME1 repeat configuration in a European family identified using a combined genomics approach.

Familial adult myoclonic epilepsy (FAME) is an adult-onset neurological disease characterized by cortical tremor, myoclonus, and seizures due to a pentanucleotide repeat expansion: a combination of pathogenic TTTCA expansion associated with a TTTTA repeat in introns of six different genes. Repeat-primed PCR (RP-PCR) is an inexpensive test for expansions at known loci. The analysis of the SAMD12 locus revealed that the repeats have different size, configuration, and composition. The TTTCA repeats can be very long (>1000 repeats) but also very short (14 being the shortest identified). Here, we report siblings of European descent with the clinical diagnosis of FAME yet a negative RP-PCR test. Using short-read genome sequencing, we identified the pentanucleotide expansion in intron 4 of SAMD12, which was confirmed by CRIPSR-Cas9-mediated enrichment and long-read sequencing to be of (TTTTA)~879 (TTTCA)3 (TTTTA)7 (TTTCA)7 configuration. Our finding is the first to associate the SAMD12 locus in European patients with FAME and currently represents the shortest identified TTTCA expansion. Our results suggest that the SAMD12 locus should be tested in patients with suspected FAME independent of ethnicity. Furthermore, RP-PCR may miss the underlying mutation, and genome sequencing may be needed to confirm the pathogenic repeat.

Polygenic risk score-based phenome-wide association study identifies novel associations for Tourette syndrome.

Tourette Syndrome (TS) is a complex neurodevelopmental disorder characterized by vocal and motor tics lasting more than a year. It is highly polygenic in nature with both rare and common previously associated variants. Epidemiological studies have shown TS to be correlated with other phenotypes, but large-scale phenome wide analyses in biobank level data have not been performed to date. In this study, we used the summary statistics from the latest meta-analysis of TS to calculate the polygenic risk score (PRS) of individuals in the UK Biobank data and applied a Phenome Wide Association Study (PheWAS) approach to determine the association of disease risk with a wide range of phenotypes. A total of 57 traits were found to be significantly associated with TS polygenic risk, including multiple psychosocial factors and mental health conditions such as anxiety disorder and depression. Additional associations were observed with complex non-psychiatric disorders such as Type 2 diabetes, heart palpitations, and respiratory conditions. Cross-disorder comparisons of phenotypic associations with genetic risk for other childhood-onset disorders (e.g.: attention deficit hyperactivity disorder [ADHD], autism spectrum disorder [ASD], and obsessive-compulsive disorder [OCD]) indicated an overlap in associations between TS and these disorders. ADHD and ASD had a similar direction of effect with TS while OCD had an opposite direction of effect for all traits except mental health factors. Sex-specific PheWAS analysis identified differences in the associations with TS genetic risk between males and females. Type 2 diabetes and heart palpitations were significantly associated with TS risk in males but not in females, whereas diseases of the respiratory system were associated with TS risk in females but not in males. This analysis provides further evidence of shared genetic and phenotypic architecture of different complex disorders.

Gain-of-function variants in the ion channel gene TRPM3 underlie a spectrum of neurodevelopmental disorders.

TRPM3 is a temperature- and neurosteroid-sensitive plasma membrane cation channel expressed in a variety of neuronal and non-neuronal cells. Recently, rare de novo variants in TRPM3 were identified in individuals with developmental and epileptic encephalopathy, but the link between TRPM3 activity and neuronal disease remains poorly understood. We previously reported that two disease-associated variants in TRPM3 lead to a gain of channel function . Here, we report a further 10 patients carrying one of seven additional heterozygous TRPM3 missense variants. These patients present with a broad spectrum of neurodevelopmental symptoms, including global developmental delay, intellectual disability, epilepsy, musculo-skeletal anomalies, and altered pain perception. We describe a cerebellar phenotype with ataxia or severe hypotonia, nystagmus, and cerebellar atrophy in more than half of the patients. All disease-associated variants exhibited a robust gain-of-function phenotype, characterized by increased basal activity leading to cellular calcium overload and by enhanced responses to the neurosteroid ligand pregnenolone sulfate when co-expressed with wild-type TRPM3 in mammalian cells. The antiseizure medication primidone, a known TRPM3 antagonist, reduced the increased basal activity of all mutant channels. These findings establish gain-of-function of TRPM3 as the cause of a spectrum of autosomal dominant neurodevelopmental disorders with frequent cerebellar involvement in humans and provide support for the evaluation of TRPM3 antagonists as a potential therapy.

Insights into familial adult myoclonus epilepsy pathogenesis: How the same repeat expansion in six unrelated genes may lead to cortical excitability.

Familial adult myoclonus epilepsy (FAME) results from the same pathogenic TTTTA/TTTCA pentanucleotide repeat expansion in six distinct genes encoding proteins with different subcellular localizations and very different functions, which poses the issue of what causes the neurobiological disturbances that lead to the clinical phenotype. Postmortem and electrophysiological studies have pointed to cortical hyperexcitability as well as dysfunction and neurodegeneration of both the cortex and cerebellum of FAME subjects. FAME expansions, contrary to the same expansion in DAB1 causing spinocerebellar ataxia type 37, seem to have no or limited impact on their recipient gene expression, which suggests a pathophysiological mechanism independent of the gene and its function. Current hypotheses include toxicity of the RNA molecules carrying UUUCA repeats, or toxicity of polypeptides encoded by the repeats, a mechanism known as repeat-associated non-AUG translation. The analysis of postmortem brains of FAME1 expansion (in SAMD12) carriers has revealed the presence of RNA foci that could be formed by the aggregation of RNA molecules with abnormal UUUCA repeats, but evidence is still lacking for other FAME subtypes. Even when the expansion is located in a gene ubiquitously expressed, expression of repeats remains undetectable in peripheral tissues (blood, skin). Therefore, the development of appropriate cellular models (induced pluripotent stem cell-derived neurons) or the study of affected tissues in patients is required to elucidate how FAME repeat expansions located in unrelated genes lead to disease.

Mosaic divergent repeat interruptions in XDP influence repeat stability and disease onset.

While many genetic causes of movement disorders have been identified, modifiers of disease expression are largely unknown. X-linked dystonia-parkinsonism (XDP) is a neurodegenerative disease caused by a SINE-VNTR-Alu(AGAGGG)n retrotransposon insertion in TAF1, with a polymorphic (AGAGGG)n repeat. Repeat length and variants in MSH3 and PMS2 explain ∼65% of the variance in age at onset (AAO) in XDP. However, additional genetic modifiers are conceivably at play in XDP, such as repeat interruptions. Long-read nanopore sequencing of PCR amplicons from XDP patients (n = 202) was performed to assess potential repeat interruption and instability. Repeat-primed PCR and Cas9-mediated targeted enrichment confirmed the presence of identified divergent repeat motifs. In addition to the canonical pure SINE-VNTR-Alu-5'-(AGAGGG)n, we observed a mosaic of divergent repeat motifs that polarized at the beginning of the tract, where the divergent repeat interruptions varied in motif length by having one, two, or three nucleotides fewer than the hexameric motif, distinct from interruptions in other disease-associated repeats, which match the lengths of the canonical motifs. All divergent configurations occurred mosaically and in two investigated brain regions (basal ganglia, cerebellum) and in blood-derived DNA from the same patient. The most common divergent interruption was AGG [5'-SINE-VNTR-Alu(AGAGGG)2AGG(AGAGGG)n], similar to the pure tract, followed by AGGG [5'-SINE-VNTR-Alu(AGAGGG)2AGGG(AGAGGG)n], at median frequencies of 0.425 (IQR: 0.42-0.43) and 0.128 (IQR: 0.12-0.13), respectively. The mosaic AGG motif was not associated with repeat number (estimate = -3.8342, P = 0.869). The mosaic pure tract frequency was associated with repeat number (estimate = 45.32, P = 0.0441) but not AAO (estimate = -41.486, P = 0.378). Importantly, the mosaic frequency of the AGGG negatively correlated with repeat number after adjusting for age at sampling (estimate = -161.09, P = 3.44 × 10-5). When including the XDP-relevant MSH3/PMS2 modifier single nucleotide polymorphisms into the model, the mosaic AGGG frequency was associated with AAO (estimate = 155.1063, P = 0.047); however, the association dissipated after including the repeat number (estimate = -92.46430, P = 0.079). We reveal novel mosaic divergent repeat interruptions affecting both motif length and sequence (DRILS) of the canonical motif polarized within the SINE-VNTR-Alu(AGAGGG)n repeat. Our study illustrates: (i) the importance of somatic mosaic genotypes; (ii) the biological plausibility of multiple modifiers (both germline and somatic) that can have additive effects on repeat instability; and (iii) that these variations may remain undetected without assessment of single molecules.