A study concept of expeditious clinical enrollment for genetic modifier studies in Charcot-Marie-Tooth neuropathy 1A.

BACKGROUND: Caused by duplications of the gene encoding peripheral myelin protein 22 (PMP22), Charcot-Marie-Tooth disease type 1A (CMT1A) is the most common hereditary neuropathy. Despite this shared genetic origin, there is considerable variability in clinical severity. It is hypothesized that genetic modifiers contribute to this heterogeneity, the identification of which may reveal novel therapeutic targets. In this study, we present a comprehensive analysis of clinical examination results from 1564 CMT1A patients sourced from a prospective natural history study conducted by the RDCRN-INC (Inherited Neuropathy Consortium). Our primary objective is to delineate extreme phenotype profiles (mild and severe) within this patient cohort, thereby enhancing our ability to detect genetic modifiers with large effects. METHODS: We have conducted large-scale statistical analyses of the RDCRN-INC database to characterize CMT1A severity across multiple metrics. RESULTS: We defined patients below the 10th (mild) and above the 90th (severe) percentiles of age-normalized disease severity based on the CMT Examination Score V2 and foot dorsiflexion strength (MRC scale). Based on extreme phenotype categories, we defined a statistically justified recruitment strategy, which we propose to use in future modifier studies. INTERPRETATION: Leveraging whole genome sequencing with base pair resolution, a future genetic modifier evaluation will include single nucleotide association, gene burden tests, and structural variant analysis. The present work not only provides insight into the severity and course of CMT1A, but also elucidates the statistical foundation and practical considerations for a cost-efficient and straightforward patient enrollment strategy that we intend to conduct on additional patients recruited globally.

Heterozygous APTX mutation associated with atypical multiple system atrophy-like phenotype: A case report.

We describe here a 73-year-old patient presenting with atypical MSA-P-like phenotype carrying a monoallelic p. W279X mutation in the APTX gene, which causes ataxia with oculomotor apraxia type 1 (AOA1) when in homozygous state. We hypothesize that rare monoallelic APTX variants could modulate MSA risk and phenotype.

Whole genome sequencing increases the diagnostic rate in Charcot-Marie-Tooth disease.

Charcot-Marie-Tooth disease (CMT) is one of the most common and genetically heterogeneous inherited neurological diseases, with more than 130 disease-causing genes. Whole genome sequencing (WGS) has improved diagnosis across genetic diseases, but the diagnostic impact in CMT is yet to be fully reported. We present the diagnostic results from a single specialist inherited neuropathy centre, including the impact of WGS diagnostic testing. Patients were assessed at our specialist inherited neuropathy centre from 2009-2023. Genetic testing was performed using single gene testing, next-generation sequencing targeted panels, research whole exome and whole genome sequencing (WGS), and latterly WGS through the UK National Health Service. Variants were assessed using the American College of Medical Genetics and Genomics and Association for Clinical Genomic Science criteria. Excluding patients with hereditary ATTR amyloidosis, 1515 patients with a clinical diagnosis of CMT and related disorders were recruited. 621 patients had CMT1 (41.0%), 294 CMT2 (19.4%), 205 intermediate CMT (CMTi, 13.5%), 139 hereditary motor neuropathy (HMN, 9.2%), 93 hereditary sensory neuropathy (HSN, 6.1%), 38 sensory ataxic neuropathy (2.5%), 72 hereditary neuropathy with liability to pressure palsies (HNPP, 4.8%) and 53 'complex' neuropathy (3.5%). Overall, a genetic diagnosis was reached in 76.9% (1165/1515). A diagnosis was most likely in CMT1 (96.8%, 601/621), followed by CMTi (81.0%, 166/205) and then HSN (69.9%, 65/93). Diagnostic rates remained less than 50% in CMT2, HMN and complex neuropathies. The most common genetic diagnosis was PMP22 duplication (CMT1A; 505/1165, 43.3%), then GJB1 (CMTX1; 151/1165, 13.0%), PMP22 deletion (HNPP; 72/1165, 6.2%) and MFN2 (CMT2A; 46/1165, 3.9%). We recruited 233 cases to the UK 100,000 Genomes Project (100KGP), of which 74 (31.8%) achieved a diagnosis; 28 had been otherwise diagnosed since recruitment leaving a true diagnostic rate of WGS through the 100KGP of 19.7% (46/233). However, almost half of the solved cases (35/74) received a negative report from the study, and the diagnosis was made through our research access to the WGS data. The overall diagnostic uplift of WGS for the entire cohort was 3.5%. Our diagnostic rate is the highest reported from a single centre, and has benefitted from the use of WGS, particularly access to the raw data. However, almost one quarter of all cases remain unsolved, and a new reference genome and novel technologies will be important to narrow the 'diagnostic gap'.

Effectiveness of ixekizumab for the treatment of moderate-to-severe plaque psoriasis with involvement of difficult-to-treat areas: A 52-week multicenter retrospective study.

Biological drugs have dramatically changed the approach to treating moderate-to-severe plaque psoriasis, achieving excellent skin clearance and safety outcomes. However, the management of difficult-to-treat areas (e.g., scalp, palms/soles, nails, and genitalia) still represents a challenge in psoriasis treatment. Data in the literature on difficult-to-treat sites are limited and, frequently, no specific analysis is performed during clinical trials. We conducted a 52-week, retrospective study to evaluate the effectiveness of ixekizumab in 120 patients with moderate-to-severe plaque psoriasis of at least one difficult-to-treat area (scalp, palmoplantar surfaces, nails, and genitalia). Ninety-nine patients had scalp psoriasis, 35 had involvement of the palms or soles, 27 were affected by genital psoriasis, and 22 patients reported involvement of the nails. After 1 year of treatment, 96% of patients with scalp involvement, 95.6% of patients with palmoplantar psoriasis, 95.2% of patients with genital psoriasis, and 85% of patients with nail involvement achieved a site-specific Physician's Global Assessment of 0 or 1 (clear or almost clear). No serious adverse events were observed during the study. Our study supports the effectiveness of ixekizumab in plaque psoriasis involving difficult-to-treat sites.

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.

Serum Neurofilament Light Chain in Replication Factor Complex Subunit 1 CANVAS and Disease Spectrum.

BACKGROUND: Biallelic intronic AAGGG repeat expansions in the replication factor complex subunit 1 (RFC1) gene were identified as the leading cause of cerebellar ataxia, neuropathy, vestibular areflexia syndrome. Patients exhibit significant clinical heterogeneity and variable disease course, but no potential biomarker has been identified to date. OBJECTIVES: In this multicenter cross-sectional study, we aimed to evaluate neurofilament light (NfL) chain serum levels in a cohort of RFC1 disease patients and to correlate NfL serum concentrations with clinical phenotype and disease severity. METHODS: Sixty-one patients with genetically confirmed RFC1 disease and 48 healthy controls (HCs) were enrolled from six neurological centers. Serum NfL concentration was measured using the single molecule array assay technique. RESULTS: Serum NfL concentration was significantly higher in patients with RFC1 disease compared to age- and-sex-matched HCs (P < 0.0001). NfL level showed a moderate correlation with age in both HCs (r = 0.4353, P = 0.0020) and patients (r = 0.4092, P = 0.0011). Mean NfL concentration appeared to be significantly higher in patients with cerebellar involvement compared to patients without cerebellar dysfunction (27.88 vs. 21.84 pg/mL, P = 0.0081). The association between cerebellar involvement and NfL remained significant after controlling for age and sex (ß = 0.260, P = 0.034). CONCLUSIONS: Serum NfL levels are significantly higher in patients with RFC1 disease compared to HCs and correlate with cerebellar involvement. Longitudinal studies are warranted to assess its change over time.

Can CANVAS due to RFC1 biallelic expansions present with pure ataxia?

BACKGROUND: Biallelic expansion of AAGGG in the replication factor complex subunit 1 (RFC1) was identified as a major cause of cerebellar ataxia, neuropathy (sensory ganglionopathy, or SG) and vestibular areflexia syndrome (CANVAS). We wanted to clarify if RFC1 expansions can present with pure ataxia and if such expansions could be responsible for some cases where an alternative diagnosis had been made. METHODS: We identified patients with a combination of ataxia and SG and no other cause found, patients where an alternative diagnosis had been made, and patients with pure ataxia. Testing for RFC1 expansions was done using established methodology. RESULTS: Among 54 patients with otherwise idiopathic sporadic ataxia without SG, none was found to have RFC1 expansions. Among 38 patients with cerebellar ataxia and SG in which all other causes were excluded, 71% had RFC1 expansions. Among 27 patients with cerebellar ataxia and SG diagnosed with coeliac disease or gluten sensitivity, 15% had RFC1 expansions. CONCLUSIONS: Isolated cerebellar ataxia without SG makes the diagnosis of CANVAS due to RFC1 expansions highly improbable, but CANVAS is frequently the cause of the combination of idiopathic cerebellar ataxia with SG. It is important to screen patients diagnosed with other causes of acquired ataxia and SG as a small percentage were found to have RFC1 expansions.

Mutations in alpha-B-crystallin cause autosomal dominant axonal Charcot-Marie-Tooth disease with congenital cataracts.

BACKGROUND AND PURPOSE: Mutations in the alpha-B-crystallin (CRYAB) gene have initially been associated with myofibrillar myopathy, dilated cardiomyopathy and cataracts. For the first time, peripheral neuropathy is reported here as a novel phenotype associated with CRYAB. METHODS: Whole-exome sequencing was performed in two unrelated families with genetically unsolved axonal Charcot-Marie-Tooth disease (CMT2), assessing clinical, neurophysiological and radiological features. RESULTS: The pathogenic CRYAB variant c.358A>G;p.Arg120Gly was segregated in all affected patients from two unrelated families. The disease presented as late onset CMT2 (onset over 40 years) with distal sensory and motor impairment and congenital cataracts. Muscle involvement was probably associated in cases showing mild axial and diaphragmatic weakness. In all cases, nerve conduction studies demonstrated the presence of an axonal sensorimotor neuropathy along with chronic neurogenic changes on needle examination. DISCUSSION: In cases with late onset autosomal dominant CMT2 and congenital cataracts, it is recommended that CRYAB is considered for genetic testing. The identification of CRYAB mutations causing CMT2 further supports a continuous spectrum of expressivity, from myopathic to neuropathic and mixed forms, of a growing number of genes involved in protein degradation and chaperone-assisted autophagy.

Unraveling the genetic landscape of undiagnosed cerebellar ataxia in Brazilian patients.

INTRODUCTION: Hereditary ataxias (HAs) encompass a diverse and genetically intricate group of rare neurodegenerative disorders, presenting diagnostic challenges. Whole-exome sequencing (WES) has significantly improved diagnostic success. This study aimed to elucidate genetic causes of cerebellar ataxia within a diverse Brazilian cohort. METHODS: Biological samples were collected from individuals with sporadic or familial cerebellar ataxia, spanning various ages and phenotypes, excluding common SCAs and Friedreich ataxia. RFC1 biallelic AAGGG repeat expansion was screened in all patients. For AAGGG-negative cases, WES targeting 441 ataxia-related genes was performed, followed by ExpansionHunter analysis for repeat expansions, including the recently described GGC-ZFHX3. Variant classification adhered to ClinGen guidelines, yielding definitive or probable diagnoses. RESULTS: The study involved 76 diverse Brazilian families. 16 % received definitive diagnoses, and another 16 % received probable ones. RFC1-related ataxia was predominant, with two definitive cases, followed by KIF1A (one definitive and one probable) and SYNE-1 (two probable). Early-onset cases exhibited higher diagnostic rates. ExpansionHunter improved diagnosis by 4 %.We did not detected GGC-ZFHX3 repeat expansion in this cohort. CONCLUSION: This study highlights diagnostic complexities in cerebellar ataxia, even with advanced genetic methods. RFC1, KIF1A, and SYNE1 emerged as prevalent mutations. ZFHX3 repeat expansion seem to be rare in Brazilian population. Early-onset cases showed higher diagnostic success. WES coupled with ExpansionHunter holds promise as a primary diagnostic tool, emphasizing the need for broader NGS accessibility in Brazil.

Optical Genome Mapping for the Molecular Diagnosis of Facioscapulohumeral Muscular Dystrophy: Advancement and Challenges.

Facioscapulohumeral muscular dystrophy (FSHD) is the second most common muscular dystrophy in adults, and it is associated with local D4Z4 chromatin relaxation, mostly via the contraction of the D4Z4 macrosatellite repeat array on chromosome 4q35. In this study, we aimed to investigate the use of Optical Genome Mapping (OGM) as a diagnostic tool for testing FSHD cases from the UK and India and to compare OGM performance with that of traditional techniques such as linear gel (LGE) and Pulsed-field gel electrophoresis (PFGE) Southern blotting (SB). A total of 6 confirmed and 19 suspected FSHD samples were processed with LGE and PFGE, respectively. The same samples were run using a Saphyr Genome-Imaging Instrument (1-color), and the data were analysed using custom EnFocus FSHD analysis. OGM was able to confirm the diagnosis of FSHD1 in all FSHD1 cases positive for SB (n = 17), and D4Z4 sizing highly correlated with PFGE-SB (p < 0.001). OGM correctly identified cases with mosaicism for the repeat array contraction (n = 2) and with a duplication of the D4Z4 repeat array. OGM is a promising new technology able to unravel structural variants in the genome and seems to be a valid tool for diagnosing FSHD1.

Chronic Cough as a Genetic Neurological Disorder? Insights from Cerebellar Ataxia with Neuropathy and Vestibular Areflexia Syndrome (CANVAS).

Chronic cough is common, and in many cases unexplained or refractory to otherwise effective treatment of associated medical conditions. Cough hypersensitivity has developed as a paradigm that helps to explain clinical and research observations that frequently point towards chronic cough as a neuropathic disorder. Cerebellar ataxia with neuropathy and vestibular areflexia syndrome (CANVAS) is a recently described neurological condition whose clinical features include gait ataxia, unsteadiness, peripheral neuropathy, and autonomic dysfunction. Chronic cough is also a common feature of the syndrome, with features of hypersensitivity, often preceding core neurological symptoms by up to 30 years or more. The genetic basis in a majority of cases of CANVAS appears to be biallelic variable repeat intron expansion sequences within RFC1, a gene normally involved in the regulation of DNA replication and repair. The same polymorphism has now been identified at an increased frequency in patients with unexplained or refractory chronic cough in the absence of defining clinical features of CANVAS. This review expands on these points, aiming to increase the awareness of CANVAS amongst clinicians and researchers working with chronic cough. We discuss the implications of a link between RFC1 disease and cough. Improved understanding of CANVAS may lead to an enhanced grasp of the pathophysiology of chronic cough, and new approaches to antitussive treatments.

Optical Genome Mapping Enables Detection and Accurate Sizing of RFC1 Repeat Expansions.

A recessive Short Tandem Repeat expansion in RFC1 has been found to be associated with cerebellar ataxia, neuropathy and vestibular areflexia syndrome (CANVAS), and to be a frequent cause of late onset ataxia and sensory neuropathy. The usual procedure for sizing these expansions is based on Southern Blotting (SB), a time-consuming and a relatively imprecise technique. In this paper, we compare SB with Optical Genome Mapping (OGM), a method for detecting Structural Variants (SVs) based on the measurement of distances between fluorescently labelled probes, for the diagnosis of RFC1 CANVAS and disease spectrum. The two methods are applied to 17 CANVAS patients' blood samples and resulting sizes compared, showing a good agreement. Further, long-read sequencing is used for two patients to investigate the agreement of sizes with either SB or OGM. Our study concludes that OGM represents a viable alternative to SB, allowing for a simpler technique, a more precise sizing of the expansion and ability to expand analysis of SV in the entire genome as opposed to SB which is a locus specific method.

Anti-interleukin-17 and anti-interleukin-23 biologic drugs for genital psoriasis: a single-center retrospective comparative study.

Genital psoriasis affects 3-33% of patients with psoriasis during the course of the disease, usually leading to a severe reduction in the patient's quality of life. This study aims to retrospectively assess the effectiveness of interleukin (IL)-23 and IL-17 inhibitors in a real-life population affected by moderate-to-severe plaque psoriasis with genital involvement coming from our dermatology department. A total of 86 patients with a diagnosis of moderate-tosevere plaque psoriasis with severe genital involvement were enrolled. Patient characteristics, psoriasis area and severity index (PASI), and static physician global assessment of genitalia (sPGAG) at each visit were recorded. During the treatment, the mean PASI decreased from 12.8 to 0.63 at week 52; a PGA of 0/1 was reached by 97.40% at week 52 and by 100% of patients (37/37) at week 104. No significant differences between IL-23 and IL-17 inhibitors were observed; indeed, the bio-naïve group of patients demonstrated a superior response compared to the group of bioexperienced patients.Our findings confirmed that IL-23 and IL-17 inhibitors are safe and effective therapeutic options for the treatment of genital psoriasis.

Repeat expansions in NOP56 are a cause of spinocerebellar ataxia Type 36 in the British population.

Spinocerebellar ataxias form a clinically and genetically heterogeneous group of neurodegenerative disorders characterized by progressive cerebellar ataxia. Their prevalence varies among populations and ethnicities. Spinocerebellar ataxia 36 is caused by a GGCCTG repeat expansion in the first intron of the NOP56 gene and is characterized by late-onset ataxia, sensorineural hearing loss and upper and lower motor neuron signs, including tongue fasciculations. Spinocerebellar ataxia 36 has been described mainly in East Asian and Western European patients and was thought to be absent in the British population. Leveraging novel bioinformatic tools to detect repeat expansions from whole-genome sequencing, we analyse the NOP56 repeat in 1257 British patients with hereditary ataxia and in 7506 unrelated controls. We identify pathogenic repeat expansions in five families (seven patients), representing the first cohort of White British descent patients with spinocerebellar ataxia 36. Employing in silico approaches using whole-genome sequencing data, we found an 87 kb shared haplotype in among the affected individuals from five families around the NOP56 repeat region, although this block was also shared between several controls, suggesting that the repeat arises on a permissive haplotype. Clinically, the patients presented with slowly progressive cerebellar ataxia with a low rate of hearing loss and variable rates of motor neuron impairment. Our findings show that the NOP56 expansion causes ataxia in the British population and that spinocerebellar ataxia 36 can be suspected in patients with a late-onset, slowly progressive ataxia, even without the findings of hearing loss and tongue fasciculation.

Gallbladder Stones in Pediatric Age: An Emerging Problem: The Risk of Difficult Cholecystectomy and the Importance of a Preoperative Evaluation.

The need for cholecystectomy during pediatric age has significantly increased in the last two decades. As biliary pathology increases, the probability of complicated cholecystectomies increases too. The aim of this article is to analyze our experience with difficult laparoscopic pediatric cholecystectomy, focusing on the importance of an accurate pre-operative imaging study. We retrospectively analyzed all patients affected by cholelithiasis who underwent laparoscopic cholecystectomy at the Pediatric Surgery Department of San Camillo Forlanini hospital of Rome and Santa Maria alle Scotte University Hospital of Siena from 2017 to 2022. Demographic data, body mass index (BMI), recovery data, laboratory tests, imaging exams, surgical findings, post operative management and outcome were taken into account. Overall, 34 pediatric patients, with a mean age of 14.1 years (6-18 years) were included, with a mean BMI of 29. All patients underwent abdominal ultrasonography and a liver MRI with cholangiography (cMRI). We identified five cases as "difficult cholecystectomies". Two subtotal cholecystectomies were performed. Cholecystectomy in pediatric surgery can be difficult. The surgeon must be able to find alternative strategies to total cholecystectomy to avoid the risk of possible bile duct injury (BDI). Pre-operative imaging study trough ultrasound and especially cMRI is crucial to recognize possible difficulties and to plan the surgery.

Frequency of GAA-FGF14 Ataxia in a Large Cohort of Brazilian Patients With Unsolved Adult-Onset Cerebellar Ataxia.

Objectives: Intronic FGF14 GAA repeat expansions have recently been found to be a common cause of hereditary ataxia (GAA-FGF14 ataxia; SCA27B). The global epidemiology and regional prevalence of this newly reported disorder remain to be established. In this study, we investigated the frequency of GAA-FGF14 ataxia in a large cohort of Brazilian patients with unsolved adult-onset ataxia. Methods: We recruited 93 index patients with genetically unsolved adult-onset ataxia despite extensive genetic investigation and genotyped the FGF14 repeat locus. Patients were recruited across 4 different regions of Brazil. Results: Of the 93 index patients, 8 (9%) carried an FGF14 (GAA)≥250 expansion. The expansion was also identified in 1 affected relative. Seven patients were of European descent, 1 was of African descent, and 1was of admixed American ancestry. One patient carrying a (GAA)376 expansion developed ataxia at age 28 years, confirming that GAA-FGF14 ataxia can occur before the age of 30 years. One patient displayed episodic symptoms, while none had downbeat nystagmus. Cerebellar atrophy was observed on brain MRI in 7 of 8 patients (87%). Discussion: Our results suggest that GAA-FGF14 ataxia is a common cause of adult-onset ataxia in the Brazilian population, although larger studies are needed to fully define its epidemiology.

POPULATION FREQUENCY OF REPEAT EXPANSIONS INDICATES INCREASED DISEASE PREVALENCE ESTIMATES ACROSS DIFFERENT POPULATIONS.

Repeat expansion disorders (REDs) are a devastating group of predominantly neurological diseases. Together they are common, affecting 1 in 3,000 people worldwide with population-specific differences. However, prevalence estimates of REDs are hampered by heterogeneous clinical presentation, variable geographic distributions, and technological limitations leading to under-ascertainment. Here, leveraging whole genome sequencing data from 82,176 individuals from different populations we found an overall carrier frequency of REDs of 1 in 340 individuals. Modelling disease prevalence using genetic data, age at onset and survival, we show that REDs are up to 3-fold more prevalent than currently reported figures. While some REDs are population-specific, e.g. Huntington's disease type 2, most REDs are represented in all broad genetic ancestries, including Africans and Asians, challenging the notion that some REDs are found only in European populations. These results have worldwide implications for local and global health communities in the diagnosis and management of REDs both at local and global levels.

Normal and pathogenic variation of RFC1 repeat expansions: implications for clinical diagnosis.

Cerebellar ataxia, neuropathy and vestibular areflexia syndrome (CANVAS) is an autosomal recessive neurodegenerative disease, usually caused by biallelic AAGGG repeat expansions in RFC1. In this study, we leveraged whole genome sequencing data from nearly 10 000 individuals recruited within the Genomics England sequencing project to investigate the normal and pathogenic variation of the RFC1 repeat. We identified three novel repeat motifs, AGGGC (n = 6 from five families), AAGGC (n = 2 from one family) and AGAGG (n = 1), associated with CANVAS in the homozygous or compound heterozygous state with the common pathogenic AAGGG expansion. While AAAAG, AAAGGG and AAGAG expansions appear to be benign, we revealed a pathogenic role for large AAAGG repeat configuration expansions (n = 5). Long-read sequencing was used to characterize the entire repeat sequence, and six patients exhibited a pure AGGGC expansion, while the other patients presented complex motifs with AAGGG or AAAGG interruptions. All pathogenic motifs appeared to have arisen from a common haplotype and were predicted to form highly stable G quadruplexes, which have previously been demonstrated to affect gene transcription in other conditions. The assessment of these novel configurations is warranted in CANVAS patients with negative or inconclusive genetic testing. Particular attention should be paid to carriers of compound AAGGG/AAAGG expansions when the AAAGG motif is very large (>500 repeats) or the AAGGG motif is interrupted. Accurate sizing and full sequencing of the satellite repeat with long-read sequencing is recommended in clinically selected cases to enable accurate molecular diagnosis and counsel patients and their families.