Novel ATXN10 repeat motif patterns in Peruvian families modify disease age at onset

Novel ATXN10 repeat motif patterns in Peruvian families modify disease age at onset | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Short Report Novel ATXN10 repeat motif patterns in Peruvian families modify disease age at onset Kamilla Sedov, Carla Manrique-Enciso, Madison James Yang, Ismael Araujo-Aliaga, and 8 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-5989910/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract Objectives: Spinocerebellar ataxia type 10 (SCA10) is an autosomal-dominant disorder caused by large intronic expansions of pentanucleotide repeats in the ATXN10 gene. While various repeat motifs have been described, emerging evidence suggests that specific repeat motifs—rather than merely repeat length alone—can significantly modify disease features such as seizure prevalence and penetrance. Methods: We employed a novel multiplex 20-gene panel with Cas9-targeted, amplification-free long-read sequencing and optical genome mapping to elucidate ATXN10 repeat motif patterns and investigate potential genotype-phenotype correlations in index cases of six clinically well-characterized multigenerational SCA10 kindreds from Peru. Results: We detected ATXN10 repeat expansions ranging from 990 to 2002 pentanucleotide repeats (4.9 to 10 kb expansions) across six families. Importantly, we identified three mixed repeat motif patterns and ratios of (ATTCT)ₙ(ATTCC)ₙ, which were associated with differences in age at disease onset and anticipation. Discussion: Specific ATXN10 repeat motif patterns and (ATTCT) n (ATTCC) n motif ratios may serve as modifiers of SCA10 age at onset rather than repeat length. ATXN10 repeat composition can only be fully resolved with long-read sequencing and makes it a fundamental diagnostic for clinical practice and genetic counseling. These findings underscore the need to adapt long-read sequencing clinical workflows to fully characterize large repeat expansions at the nucleotide level. Medical Genetics Long-read sequencing LRS PureTarget Cas9 targeted sequencing amplification-free LRS optical genome mapping OGM Bionano Ataxin 10 ATXN10 spinocerebellar ataxia type 10 SCA10 Figures Figure 1 Figure 2 Introduction Spinocerebellar Ataxia Type 10 (SCA10, OMIM# 603516) is a rare autosomal-dominant disorder caused by an expanded pentanucleotide repeat in the ATXN10 gene on chromosome 22q13.3. 1 Although rare globally, SCA10 is most common among Indigenous Americans, especially in Peru, where it represents 45% of SCA10 cases in a population that is about 70% Amerindian. 2 , 3 A typical ATXN10 allele has 10–32 ATTCT repeats. Intermediate alleles from 280–850 repeats may have reduced penetrance, 4 while alleles over 850 repeats result in full disease penetrance. 5 , 6 In our recent study on ATXN10 expansions in healthy Peruvians, we found expanded alleles in 3.7% of Mestizos and 9.9% of Indigenous American non-ataxic individuals. 7 Conventional methods fail to accurately determine ATXN10 repeat size and structure: Southern blot often overestimates repeats, while repeat-primed PCR cannot measure repeats over 1,250 bp or detect alternate repeats without specific repeat primers. 8 Both optical genome mapping (OGM) and Cas9-targeted amplification-free long-read sequencing (LRS) can fully resolve multi-kilobase repeat expansions. Furthermore using LRS, we and others found that ATXN10 expansions can include complex motifs like (ATTCT)n-(ATTCC)n, (ATTCT)n-(ATCCT)n-(ATCCC)n, and other penta- or heptanucleotide repeats. 9 , 10 Certain repeat motifs (i.e. ATCCT or ATTCC) are associated with specific clinical features, such as epilepsy. Furthermore, pure ATTCT repeats can show a markedly reduced penetrance. 9 , 11 In this study, we used a novel 20-gene panel for repeat expansion disorders with Cas9-targeted amplification-free LRS and OGM to characterize the full length of the ATXN10 repeat and to reveal distinct ATXN10 repeat patterns in six Peruvian index cases of multigenerational kindreds. Methods Protocol Approvals The study was approved under IRB-016-2021-CIEI-INCN at Instituto Nacional de Ciencias Neurologicas. Stanford University received the samples de-identified and genetic analysis was deemed exempt under IRB-69900. Written informed consent was obtained from all participants in the study. PureTarget long-read sequencing (LRS) DNA extraction for LRS was performed using the Nanobind PanDNA kit (PacBio,103-260-000) from frozen blood samples following the RBC lysis protocol (PacBio, 103-377-500). Short DNA fragments were removed from the samples using the short read eliminator (SRE) kit (PacBio, 102-208-300) (Supplemental Table 1). DNA libraries were prepared following protocol PacBio, 103-329-400. Four ng of DNA was aliquoted per sample before the dephosphorylation step except for sample ID 54.01 which produced only 3 ng of DNA per reaction. Duplicates of samples were digested with Cas9 and gRNA complex with either the PureTarget 20-gene gRNA panel or a single gRNA for the ATXN10 target 12 , and further processed according to the protocol. Samples were pooled as multiples of eight, concentrated into 15 ul per 8-plex, and stored at -20C until Annealing, Binding, and SMRTbell Cleanup (ABC) for the Revio system at the Stanford Genomics Service Center. Bioinformatic analysis is described in Supplemental methods. Optical genome mapping (OGM) Ultra-high molecular weight DNA was extracted from frozen blood samples with 1.5x10^6 cells using the Bionano Prep SP-G2 Blood & Cell Culture DNA Isolation Kit (Bionano Genomics, PN 80060) and following protocol CG-00006. DNA samples were labeled according to protocol CG-30553-1. Labeled samples were loaded into chips (Bionano Genomics, PN 20440) and imaged through the Bionano Saphyr instrument, with a flowthrough target of 1500 Gbp (Supplemental Table 2). Raw molecule files were analyzed using the de novo assembly workflow of Bionano Access version 1.7.2 and hg38 reference genome. Repeat expansions were found as insertions at RefStart position chr22: 45,794,058. Results Clinical presentation of SCA10 cases and ATXN10 repeat expansions All familial index cases were clinically diagnosed with SCA10 based on comprehensive neurological assessment and ataxia motor scales (SARA and NESSCA 13 , 14 ) The age at onset was between 30 and 65 years, all cases had a family history of ataxia and presented with typical gait ataxia, dysarthria, variable oculomotor symptoms, behavioral disturbances, and seizures. A summary of key clinical features is listed in Table 1 for all individuals. Additional detailed clinical information and pedigrees for each of the index cases with SCA10 is listed in Supplemental Table 3 and Supplemental Fig. 1. Table 1 Family.individual ID 70.01 54.01 62.01 35.01 28.01 50.01 Sex female male female female male female Family history positive positive positive positive positive positive Age at onset of ataxia 30 56 35 35 41 65 Disease duration 17 4 5 30 4 11 SARA score 38 7 14 16 13 16 NESSCA score 14 10 10 13 10 8 ATXN10 expanded allele 1213 2002 1323 990 1136 1358 # ATCCT 279 1765 781 839 881 1119 # ATTCC 864 177 503 148 246 192 % ATTCT of total expansion 24.41% 90.89% 60.83% 85.01% 78.17% 85.35% We detected ATXN10 repeat expansions ranging from 990 to 2002 pentanucleotide repeats (4.9 to 10kb expansion) in the six index cases (Figs. 1 and 2 ) with some somatic variability of the repeat expansions (Supplemental Fig. 2). We compared the 20-gene panel with a single ATXN10 guide RNA and found comparable number of reads, read sizes, and somatic variability (Supplemental Table 4). OGM and LRS produced nearly the same repeat sizes, differing only by 0.75–2.98% (69 to 184 bp). OGM measurements tend to be slightly larger because the fluorescent labels are placed outside the repeat region (Supplemental Table 5). No other pathogenic repeat expansions were detected in the 20-gene panel (Supplemental Table 6). Three distinct ATXN10 repeat patterns are associated with disease onset and anticipation In the six Peruvian index cases, we detected both the common ATTCT motif and the alternate ATTCC motif. Notably as a novel finding, we identified specific mixed repeat motif patterns of (ATTCT) n (ATTCC) n or (ATTCT) n (ATTCC) n (ATTCT) n , potentially influencing age at onset and anticipation (Fig. 2 ). Comparing the ATXN10 repeat motif patterns, we identified three subtypes: Pattern A: (ATTCT) n (ATTCC) n with an ATTCT:ATTCC ratio 1, and Pattern C: (ATTCT) n (ATTCC) n (ATTCT) n where ATTCC is flanked on both sides by ATTCT (Fig. 2 ). Interestingly, Pattern A in family 70.01 presented an earlier age at onset (30 years) compared to the other families and a strong anticipation in each subsequent generation (about 10 years per generation). Pattern B showed an intermediate age at onset with an earlier ataxia onset the more ATTCC motifs were present. Lastly, Pattern C presented with an age at onset over 55 years (Fig. 2 ). Discussion Using Cas9-targeted LRS and OGM, we accurately resolved one of the largest intronic repeat expansions—the ATXN10 gene—with only minor size discrepancies between these two techniques. Crucially, Cas9-targeted LRS enabled us to precisely identify distinct repeat patterns and motifs. In 2017, we pioneered the amplification-free, Cas9-based LRS method for analyzing ATXN10 repeat expansions, which we subsequently refined into a multiplex panel 12 . In this study, we used a further improved version that multiplexes 20 genes for repeat expansion disorders (PacBio PureTarget). Our key novel finding is the prominence of the alternate ATTCC motif alongside the common ATTCT motif in these six Peruvian families. While repeat length alone does not appear to drive anticipation in SCA10 (Supplemental Fig. 3), we uncovered that the ratio of the ATTCC motif within three distinct repeat patterns correlates with both disease onset and anticipation—higher proportions of the alternate repeat motif are linked to earlier onset. These observations could help clarify previously conflicting evidence on anticipation in SCA10, which was not explained by repeat length alone. 15 Instead, our findings indicate that the composition of the repeat motif pattern could underlie variability in age at onset and anticipation. This underscores the importance of fully mapping diverse ATXN10 repeat motifs with LRS, as distinct motif patterns are increasingly associated with specific clinical outcomes. Ultimately, precise genetic characterization will guide improved clinical practice, genetic counseling, and patient and family care. Declarations Acknowledgments. We are thankful for the research participants spending their time for clinical evaluation and donating blood samples for advancing research on ATXN10 .The sequencing was funded by internal start-up funds (B.S.). This work was partially funded by CONCYTEC-PROCIENCIA (N 148-2020-FONDECYT) (M.C.-O.). We are grateful to the DNA-Neurogenetics Bank of the Instituto Nacional de Ciencias Neurológicas for supporting the collection of DNA samples and associated data use in this publication. This research was funded in whole or in part MJFF-023323 by the Michael J. Fox Foundation for Parkinson’s Research (M.C.-O.). Conflict of Interest. ED, SK, SK are employees of Pacific Biosciences. All other authors declare no conflict of interest. References Zhang N, Ashizawa T Mechanistic and Therapeutic Insights into Ataxic Disorders with Pentanucleotide Expansions. Cells 2022;11. Cornejo-Olivas M, Inca-Martinez M, Castilhos RM et al (2020) Genetic Analysis of Hereditary Ataxias in Peru Identifies SCA10 Families with Incomplete Penetrance. Cerebellum 19:208–215 Jardim LB, Hasan A, Kuo SH et al (2023) An Exploratory Survey on the Care for Ataxic Patients in the American Continents and the Caribbean. Cerebellum 22:708–718 Bushara K, Bower M, Liu J et al (2013) Expansion of the Spinocerebellar ataxia type 10 (SCA10) repeat in a patient with Sioux Native American ancestry. PLoS ONE 8:e81342 Rasmussen A, Matsuura T, Ruano L et al (2001) Clinical and genetic analysis of four Mexican families with spinocerebellar ataxia type 10. Ann Neurol 50:234–239 Teive HA, Roa BB, Raskin S et al (2004) Clinical phenotype of Brazilian families with spinocerebellar ataxia 10. Neurology 63:1509–1512 Milla-Neyra K, Araujo-Aliaga I, Manrique-Enciso C et al (2025) Novel expanded ATXN10 alleles in the healthy Peruvian population: a matter of Indigenous American ethnic origin. Cerebellum (Accepted) Hashem V, Tiwari A, Bewick B et al (2020) Pulse-Field capillary electrophoresis of repeat-primed PCR amplicons for analysis of large repeats in Spinocerebellar Ataxia Type 10. PLoS ONE 15:e0228789 Schüle B, McFarland KN, Lee K et al (2017) Parkinson's disease associated with pure ATXN10 repeat expansion. NPJ Parkinsons Dis 3:27 McFarland KN, Liu J, Landrian I et al (2015) SMRT Sequencing of Long Tandem Nucleotide Repeats in SCA10 Reveals Unique Insight of Repeat Expansion Structure. PLoS ONE 10:e0135906 Morato Torres CA, Zafar F, Tsai YC et al (2022) ATTCT and ATTCC repeat expansions in the ATXN10 gene affect disease penetrance of spinocerebellar ataxia type 10. HGG Adv 3:100137 Tsai Y-C, Zafar F, McEachin ZT et al (2022) Multiplex CRISPR/Cas9-Guided No-Amp Targeted Sequencing Panel for Spinocerebellar Ataxia Repeat Expansions. In: Proukakis C (ed) Genomic Structural Variants in Nervous System Disorders. Springer US, New York, NY, pp 95–120 Schmitz-Hübsch T, du Montcel ST, Baliko L et al (2006) Scale for the assessment and rating of ataxia: development of a new clinical scale. Neurology 66:1717–1720 Kieling C, Rieder CR, Silva AC et al (2008) A neurological examination score for the assessment of spinocerebellar ataxia 3 (SCA3). Eur J Neurol 15:371–376 McFarland KN, Liu J, Landrian I et al (2013) Paradoxical effects of repeat interruptions on spinocerebellar ataxia type 10 expansions and repeat instability. Eur J Hum Genet. ;21:1272–1276. Additional Declarations The authors declare potential competing interests as follows: ED, SK, SK are employees of Pacific Biosciences. All other authors declare no conflict of interest. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-5989910","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Short Report","associatedPublications":[],"authors":[{"id":413072482,"identity":"9351ef01-1e3e-4522-8eff-7b83301acb7e","order_by":0,"name":"Kamilla Sedov","email":"","orcid":"https://orcid.org/0009-0006-3241-5165","institution":"Department of Pathology, Stanford University School of Medicine, California, USA","correspondingAuthor":false,"prefix":"","firstName":"Kamilla","middleName":"","lastName":"Sedov","suffix":""},{"id":413072483,"identity":"ade86130-281b-4797-9dff-c17120acce94","order_by":1,"name":"Carla 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22:54:04","currentVersionCode":1,"declarations":{"humanSubjects":true,"vertebrateSubjects":false,"conflictsOfInterestStatement":true,"humanSubjectEthicalGuidelines":true,"humanSubjectConsent":true,"humanSubjectClinicalTrial":false,"humanSubjectCaseReport":false,"vertebrateSubjectEthicalGuidelines":false},"doi":"10.21203/rs.3.rs-5989910/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-5989910/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":76108300,"identity":"61eaf033-44d0-47b1-9dc4-79e47367cabe","added_by":"auto","created_at":"2025-02-12 11:35:22","extension":"jpg","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":284585,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eVisualization of repeat expansion in ATXN10 with optical genome mapping. \u003c/strong\u003eOptical genome mapping identifies repeat expansions as insertions. The green horizontal bar represents reference genome (hg38) with the dark blue vertical marks corresponding to CTTAAG motif occurring every ~5kbp and recognized by the labeling enzyme DLE-1. The blue horizontal bar represents a sample genome for each of the six cases. Genome maps are produced by taking high resolution images of labeled and stained ultra-high molecular weight DNA (\u0026gt;150 kbp) that are aligned to the reference genome. An expansion is represented by a blue trapezoid figure showing an increase of the distance between the two labels on the blue genome map and is supported by molecules imaged below as blue lines. ATXN10 gene is shown as yellow horizontal line above the reference genome and exon 10 is shown as a thick vertical mark.\u003c/p\u003e","description":"","filename":"1.jpg","url":"https://assets-eu.researchsquare.com/files/rs-5989910/v1/a5779cee3647bc07cc3e158b.jpg"},{"id":76106248,"identity":"f9d10df2-b489-416a-ba0e-99223a310a5e","added_by":"auto","created_at":"2025-02-12 11:19:22","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":525400,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eCas9-targeted LRS sequencing reveals three distinct motif patterns. \u003c/strong\u003eThe ATXN10 repeat expansion motifs depicted as waterfall plots are color coded in blue = ATTCT and purple = ATTCC. Age at ataxia onset, ATTCT content, motif counts, length, and anticipation are listed\u003c/p\u003e","description":"","filename":"2.png","url":"https://assets-eu.researchsquare.com/files/rs-5989910/v1/07e92058c19efc723c9fc27f.png"},{"id":76108311,"identity":"d0a93cc6-0fad-4f7d-90eb-b549cf68b177","added_by":"auto","created_at":"2025-02-12 11:35:29","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":1452483,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-5989910/v1/1ad3e91d-26ba-458a-a621-ad1c92c3692d.pdf"},{"id":76106249,"identity":"268e57e9-e4b8-480a-9de7-3e21cf817aa6","added_by":"auto","created_at":"2025-02-12 11:19:22","extension":"docx","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":2271780,"visible":true,"origin":"","legend":"","description":"","filename":"Supplementalmaterials.docx","url":"https://assets-eu.researchsquare.com/files/rs-5989910/v1/9232e1db8339f712e5abd250.docx"}],"financialInterests":"The authors declare potential competing interests as follows: ED, SK, SK are employees of Pacific Biosciences. All other authors declare no conflict of interest.","formattedTitle":"\u003cp\u003e\u003cstrong\u003eNovel \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003eATXN10\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e repeat motif patterns in Peruvian families modify disease age at onset\u003c/strong\u003e\u003c/p\u003e","fulltext":[{"header":"Introduction","content":"\u003cp\u003eSpinocerebellar Ataxia Type 10 (SCA10, OMIM# 603516) is a rare autosomal-dominant disorder caused by an expanded pentanucleotide repeat in the \u003cem\u003eATXN10\u003c/em\u003e gene on chromosome 22q13.3. \u003csup\u003e\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e\u003c/sup\u003e Although rare globally, SCA10 is most common among Indigenous Americans, especially in Peru, where it represents 45% of SCA10 cases in a population that is about 70% Amerindian. \u003csup\u003e\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e2\u003c/span\u003e, \u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e\u003c/sup\u003e\u003c/p\u003e \u003cp\u003eA typical \u003cem\u003eATXN10\u003c/em\u003e allele has 10\u0026ndash;32 ATTCT repeats. Intermediate alleles from 280\u0026ndash;850 repeats may have reduced penetrance, \u003csup\u003e\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e\u003c/sup\u003e while alleles over 850 repeats result in full disease penetrance. \u003csup\u003e\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e, \u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e\u003c/sup\u003e In our recent study on \u003cem\u003eATXN10\u003c/em\u003e expansions in healthy Peruvians, we found expanded alleles in 3.7% of Mestizos and 9.9% of Indigenous American non-ataxic individuals. \u003csup\u003e\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e\u003c/sup\u003e\u003c/p\u003e \u003cp\u003eConventional methods fail to accurately determine \u003cem\u003eATXN10\u003c/em\u003e repeat size and structure: Southern blot often overestimates repeats, while repeat-primed PCR cannot measure repeats over 1,250 bp or detect alternate repeats without specific repeat primers. \u003csup\u003e\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e\u003c/sup\u003e\u003c/p\u003e \u003cp\u003eBoth optical genome mapping (OGM) and Cas9-targeted amplification-free long-read sequencing (LRS) can fully resolve multi-kilobase repeat expansions. Furthermore using LRS, we and others found that \u003cem\u003eATXN10\u003c/em\u003e expansions can include complex motifs like (ATTCT)n-(ATTCC)n, (ATTCT)n-(ATCCT)n-(ATCCC)n, and other penta- or heptanucleotide repeats. \u003csup\u003e\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e, \u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e\u003c/sup\u003e Certain repeat motifs (i.e. ATCCT or ATTCC) are associated with specific clinical features, such as epilepsy. Furthermore, pure ATTCT repeats can show a markedly reduced penetrance. \u003csup\u003e\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e, \u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e\u003c/sup\u003e\u003c/p\u003e \u003cp\u003eIn this study, we used a novel 20-gene panel for repeat expansion disorders with Cas9-targeted amplification-free LRS and OGM to characterize the full length of the \u003cem\u003eATXN10\u003c/em\u003e repeat and to reveal distinct \u003cem\u003eATXN10\u003c/em\u003e repeat patterns in six Peruvian index cases of multigenerational kindreds.\u003c/p\u003e"},{"header":"Methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003eProtocol Approvals\u003c/h2\u003e \u003cp\u003eThe study was approved under IRB-016-2021-CIEI-INCN at Instituto Nacional de Ciencias Neurologicas. Stanford University received the samples de-identified and genetic analysis was deemed exempt under IRB-69900. Written informed consent was obtained from all participants in the study.\u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003ePureTarget long-read sequencing (LRS)\u003c/h3\u003e\n\u003cp\u003eDNA extraction for LRS was performed using the Nanobind PanDNA kit (PacBio,103-260-000) from frozen blood samples following the RBC lysis protocol (PacBio, 103-377-500). Short DNA fragments were removed from the samples using the short read eliminator (SRE) kit (PacBio, 102-208-300) (Supplemental Table\u0026nbsp;1). DNA libraries were prepared following protocol PacBio, 103-329-400. Four ng of DNA was aliquoted per sample before the dephosphorylation step except for sample ID 54.01 which produced only 3 ng of DNA per reaction. Duplicates of samples were digested with Cas9 and gRNA complex with either the PureTarget 20-gene gRNA panel or a single gRNA for the \u003cem\u003eATXN10\u003c/em\u003e target\u003csup\u003e\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e\u003c/sup\u003e, and further processed according to the protocol. Samples were pooled as multiples of eight, concentrated into 15 ul per 8-plex, and stored at -20C until Annealing, Binding, and SMRTbell Cleanup (ABC) for the Revio system at the Stanford Genomics Service Center. Bioinformatic analysis is described in Supplemental methods.\u003c/p\u003e\n\u003ch3\u003eOptical genome mapping (OGM)\u003c/h3\u003e\n\u003cp\u003eUltra-high molecular weight DNA was extracted from frozen blood samples with 1.5x10^6 cells using the Bionano Prep SP-G2 Blood \u0026amp; Cell Culture DNA Isolation Kit (Bionano Genomics, PN 80060) and following protocol CG-00006. DNA samples were labeled according to protocol CG-30553-1. Labeled samples were loaded into chips (Bionano Genomics, PN 20440) and imaged through the Bionano Saphyr instrument, with a flowthrough target of 1500 Gbp (Supplemental Table\u0026nbsp;2). Raw molecule files were analyzed using the \u003cem\u003ede novo\u003c/em\u003e assembly workflow of Bionano Access version 1.7.2 and hg38 reference genome. Repeat expansions were found as insertions at RefStart position chr22: 45,794,058.\u003c/p\u003e"},{"header":"Results","content":"\u003cp\u003e \u003cb\u003eClinical presentation of SCA10 cases and\u003c/b\u003e \u003cb\u003eATXN10\u003c/b\u003e \u003cb\u003erepeat expansions\u003c/b\u003e\u003c/p\u003e \u003cp\u003eAll familial index cases were clinically diagnosed with SCA10 based on comprehensive neurological assessment and ataxia motor scales (SARA and NESSCA\u003csup\u003e\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e, \u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e\u003c/sup\u003e) The age at onset was between 30 and 65 years, all cases had a family history of ataxia and presented with typical gait ataxia, dysarthria, variable oculomotor symptoms, behavioral disturbances, and seizures. A summary of key clinical features is listed in Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e for all individuals. Additional detailed clinical information and pedigrees for each of the index cases with SCA10 is listed in Supplemental Table\u0026nbsp;3 and Supplemental Fig.\u0026nbsp;1.\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab1\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 1\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e\u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"7\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c6\" colnum=\"6\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c7\" colnum=\"7\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003eFamily.individual ID\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003e70.01\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003e54.01\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e \u003cp\u003e62.01\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c5\"\u003e \u003cp\u003e35.01\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c6\"\u003e \u003cp\u003e28.01\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c7\"\u003e \u003cp\u003e50.01\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eSex\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003efemale\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003emale\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003efemale\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003efemale\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003emale\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003efemale\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eFamily history\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003epositive\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003epositive\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003epositive\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003epositive\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003epositive\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003epositive\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eAge at onset of ataxia\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e30\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e56\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e35\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e35\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e41\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e65\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eDisease duration\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e17\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e4\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e5\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e30\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e4\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e11\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eSARA score\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e38\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e7\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e14\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e16\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e13\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e16\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eNESSCA score\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e14\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e10\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e10\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e13\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e10\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e8\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eATXN10 expanded allele\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e1213\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e2002\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e1323\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e990\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e1136\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e1358\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003e# ATCCT\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e279\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e1765\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e781\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e839\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e881\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e1119\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003e# ATTCC\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e864\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e177\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e503\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e148\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e246\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e192\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003e% ATTCT of total expansion\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e24.41%\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e90.89%\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e60.83%\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e85.01%\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e78.17%\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e85.35%\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003cp\u003eWe detected \u003cem\u003eATXN10\u003c/em\u003e repeat expansions ranging from 990 to 2002 pentanucleotide repeats (4.9 to 10kb expansion) in the six index cases (Figs.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e and \u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e) with some somatic variability of the repeat expansions (Supplemental Fig.\u0026nbsp;2). We compared the 20-gene panel with a single ATXN10 guide RNA and found comparable number of reads, read sizes, and somatic variability (Supplemental Table\u0026nbsp;4). OGM and LRS produced nearly the same repeat sizes, differing only by 0.75\u0026ndash;2.98% (69 to 184 bp). OGM measurements tend to be slightly larger because the fluorescent labels are placed outside the repeat region (Supplemental Table\u0026nbsp;5). No other pathogenic repeat expansions were detected in the 20-gene panel (Supplemental Table\u0026nbsp;6).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003cb\u003eThree distinct\u003c/b\u003e \u003cb\u003eATXN10\u003c/b\u003e \u003cb\u003erepeat patterns are associated with disease onset and anticipation\u003c/b\u003e\u003c/p\u003e \u003cp\u003eIn the six Peruvian index cases, we detected both the common ATTCT motif and the alternate ATTCC motif. Notably as a novel finding, we identified specific mixed repeat motif patterns of (ATTCT)\u003csub\u003en\u003c/sub\u003e(ATTCC)\u003csub\u003en\u003c/sub\u003e or (ATTCT)\u003csub\u003en\u003c/sub\u003e(ATTCC)\u003csub\u003en\u003c/sub\u003e(ATTCT)\u003csub\u003en\u003c/sub\u003e, potentially influencing age at onset and anticipation (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e). Comparing the \u003cem\u003eATXN10\u003c/em\u003e repeat motif patterns, we identified three subtypes: Pattern A: (ATTCT) \u003csub\u003en\u003c/sub\u003e (ATTCC) \u003csub\u003en\u003c/sub\u003e with an ATTCT:ATTCC ratio\u0026thinsp;\u0026lt;\u0026thinsp;1, Pattern B: (ATTCT) \u003csub\u003en\u003c/sub\u003e (ATTCC) \u003csub\u003en\u003c/sub\u003e with an ATTCT:ATTCC ratio\u0026thinsp;\u0026gt;\u0026thinsp;1, and Pattern C: (ATTCT) \u003csub\u003en\u003c/sub\u003e (ATTCC) \u003csub\u003en\u003c/sub\u003e (ATTCT) \u003csub\u003en\u003c/sub\u003e where ATTCC is flanked on both sides by ATTCT (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e). Interestingly, Pattern A in family 70.01 presented an earlier age at onset (30 years) compared to the other families and a strong anticipation in each subsequent generation (about 10 years per generation). Pattern B showed an intermediate age at onset with an earlier ataxia onset the more ATTCC motifs were present. Lastly, Pattern C presented with an age at onset over 55 years (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e).\u003c/p\u003e"},{"header":"Discussion","content":"\u003cp\u003eUsing Cas9-targeted LRS and OGM, we accurately resolved one of the largest intronic repeat expansions\u0026mdash;the \u003cem\u003eATXN10\u003c/em\u003e gene\u0026mdash;with only minor size discrepancies between these two techniques.\u003c/p\u003e \u003cp\u003eCrucially, Cas9-targeted LRS enabled us to precisely identify distinct repeat patterns and motifs. In 2017, we pioneered the amplification-free, Cas9-based LRS method for analyzing \u003cem\u003eATXN10\u003c/em\u003e repeat expansions, which we subsequently refined into a multiplex panel\u003csup\u003e\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e\u003c/sup\u003e. In this study, we used a further improved version that multiplexes 20 genes for repeat expansion disorders (PacBio PureTarget).\u003c/p\u003e \u003cp\u003eOur key novel finding is the prominence of the alternate ATTCC motif alongside the common ATTCT motif in these six Peruvian families. While repeat length alone does not appear to drive anticipation in SCA10 (Supplemental Fig.\u0026nbsp;3), we uncovered that the ratio of the ATTCC motif within three distinct repeat patterns correlates with both disease onset and anticipation\u0026mdash;higher proportions of the alternate repeat motif are linked to earlier onset.\u003c/p\u003e \u003cp\u003eThese observations could help clarify previously conflicting evidence on anticipation in SCA10, which was not explained by repeat length alone. \u003csup\u003e\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e\u003c/sup\u003e Instead, our findings indicate that the composition of the repeat motif pattern could underlie variability in age at onset and anticipation. This underscores the importance of fully mapping diverse ATXN10 repeat motifs with LRS, as distinct motif patterns are increasingly associated with specific clinical outcomes. Ultimately, precise genetic characterization will guide improved clinical practice, genetic counseling, and patient and family care.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eAcknowledgments.\u0026nbsp;\u003c/strong\u003eWe are thankful for the research participants spending their time for clinical evaluation and donating blood samples for advancing research on \u003cem\u003eATXN10\u003c/em\u003e.The sequencing was funded by internal start-up funds (B.S.). This work was partially funded by CONCYTEC-PROCIENCIA (N 148-2020-FONDECYT) (M.C.-O.). We are grateful to the DNA-Neurogenetics Bank of the Instituto Nacional de Ciencias Neurol\u0026oacute;gicas for supporting the collection of DNA samples and associated data use in this publication. This research was funded in whole or in part MJFF-023323 by the Michael J. Fox Foundation for Parkinson\u0026rsquo;s Research (M.C.-O.).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConflict of Interest.\u0026nbsp;\u003c/strong\u003eED, SK, SK are employees of Pacific Biosciences. All other authors declare no conflict of interest.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eZhang N, Ashizawa T Mechanistic and Therapeutic Insights into Ataxic Disorders with Pentanucleotide Expansions. Cells 2022;11.\u003c/li\u003e\n\u003cli\u003eCornejo-Olivas M, Inca-Martinez M, Castilhos RM et al (2020) Genetic Analysis of Hereditary Ataxias in Peru Identifies SCA10 Families with Incomplete Penetrance. Cerebellum 19:208\u0026ndash;215\u003c/li\u003e\n\u003cli\u003eJardim LB, Hasan A, Kuo SH et al (2023) An Exploratory Survey on the Care for Ataxic Patients in the American Continents and the Caribbean. Cerebellum 22:708\u0026ndash;718\u003c/li\u003e\n\u003cli\u003eBushara K, Bower M, Liu J et al (2013) Expansion of the Spinocerebellar ataxia type 10 (SCA10) repeat in a patient with Sioux Native American ancestry. PLoS ONE 8:e81342\u003c/li\u003e\n\u003cli\u003eRasmussen A, Matsuura T, Ruano L et al (2001) Clinical and genetic analysis of four Mexican families with spinocerebellar ataxia type 10. Ann Neurol 50:234\u0026ndash;239\u003c/li\u003e\n\u003cli\u003eTeive HA, Roa BB, Raskin S et al (2004) Clinical phenotype of Brazilian families with spinocerebellar ataxia 10. Neurology 63:1509\u0026ndash;1512\u003c/li\u003e\n\u003cli\u003eMilla-Neyra K, Araujo-Aliaga I, Manrique-Enciso C et al (2025) Novel expanded ATXN10 alleles in the healthy Peruvian population: a matter of Indigenous American ethnic origin. Cerebellum (Accepted)\u003c/li\u003e\n\u003cli\u003eHashem V, Tiwari A, Bewick B et al (2020) Pulse-Field capillary electrophoresis of repeat-primed PCR amplicons for analysis of large repeats in Spinocerebellar Ataxia Type 10. PLoS ONE 15:e0228789\u003c/li\u003e\n\u003cli\u003eSch\u0026uuml;le B, McFarland KN, Lee K et al (2017) Parkinson's disease associated with pure ATXN10 repeat expansion. NPJ Parkinsons Dis 3:27\u003c/li\u003e\n\u003cli\u003eMcFarland KN, Liu J, Landrian I et al (2015) SMRT Sequencing of Long Tandem Nucleotide Repeats in SCA10 Reveals Unique Insight of Repeat Expansion Structure. PLoS ONE 10:e0135906\u003c/li\u003e\n\u003cli\u003eMorato Torres CA, Zafar F, Tsai YC et al (2022) ATTCT and ATTCC repeat expansions in the ATXN10 gene affect disease penetrance of spinocerebellar ataxia type 10. HGG Adv 3:100137\u003c/li\u003e\n\u003cli\u003eTsai Y-C, Zafar F, McEachin ZT et al (2022) Multiplex CRISPR/Cas9-Guided No-Amp Targeted Sequencing Panel for Spinocerebellar Ataxia Repeat Expansions. In: Proukakis C (ed) Genomic Structural Variants in Nervous System Disorders. Springer US, New York, NY, pp 95\u0026ndash;120\u003c/li\u003e\n\u003cli\u003eSchmitz-H\u0026uuml;bsch T, du Montcel ST, Baliko L et al (2006) Scale for the assessment and rating of ataxia: development of a new clinical scale. Neurology 66:1717\u0026ndash;1720\u003c/li\u003e\n\u003cli\u003eKieling C, Rieder CR, Silva AC et al (2008) A neurological examination score for the assessment of spinocerebellar ataxia 3 (SCA3). Eur J Neurol 15:371\u0026ndash;376\u003c/li\u003e\n\u003cli\u003eMcFarland KN, Liu J, Landrian I et al (2013) Paradoxical effects of repeat interruptions on spinocerebellar ataxia type 10 expansions and repeat instability. Eur J Hum Genet. ;21:1272\u0026ndash;1276.\u0026nbsp;\u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[{"identity":"54359de0-c83f-49e9-bcf1-b4a645f20e2a","identifier":"10.13039/100000864","name":"Michael J. Fox Foundation for Parkinson's Research","awardNumber":"MJFF-023323 ","order_by":0},{"identity":"1f46ed74-a021-4f5f-bdde-53da4961824e","identifier":"10.13039/501100010747","name":"Consejo Nacional de Ciencia, Tecnología e Innovación Tecnológica","awardNumber":"N 148-2020-FONDECYT","order_by":1}],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":true,"hideJournal":true,"highlight":"","institution":"Stanford University School of Medicine","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"Long-read sequencing, LRS, PureTarget, Cas9 targeted sequencing, amplification-free LRS, optical genome mapping, OGM, Bionano, Ataxin 10, ATXN10, spinocerebellar ataxia type 10, SCA10","lastPublishedDoi":"10.21203/rs.3.rs-5989910/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-5989910/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003e\u003cstrong\u003eObjectives: \u003c/strong\u003eSpinocerebellar ataxia type 10 (SCA10) is an autosomal-dominant disorder caused by large intronic expansions of pentanucleotide repeats in the \u003cem\u003eATXN10\u003c/em\u003e gene. While various repeat motifs have been described, emerging evidence suggests that specific repeat motifs—rather than merely repeat length alone—can significantly modify disease features such as seizure prevalence and penetrance.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eMethods: \u003c/strong\u003eWe employed a novel multiplex 20-gene panel with Cas9-targeted, amplification-free long-read sequencing and optical genome mapping to elucidate ATXN10 repeat motif patterns and investigate potential genotype-phenotype correlations in index cases of six clinically well-characterized multigenerational SCA10 kindreds from Peru.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eResults: \u003c/strong\u003eWe detected \u003cem\u003eATXN10\u003c/em\u003e repeat expansions ranging from 990 to 2002 pentanucleotide repeats (4.9 to 10 kb expansions) across six families. Importantly, we identified three mixed repeat motif patterns and ratios of (ATTCT)ₙ(ATTCC)ₙ, which were associated with differences in age at disease onset and anticipation.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eDiscussion: \u003c/strong\u003eSpecific \u003cem\u003eATXN10\u003c/em\u003e repeat motif patterns and (ATTCT)\u003csub\u003en\u003c/sub\u003e(ATTCC)\u003csub\u003en\u003c/sub\u003e motif ratios may serve as modifiers of SCA10 age at onset rather than repeat length. \u003cem\u003eATXN10\u003c/em\u003e repeat composition can only be fully resolved with long-read sequencing and makes it a fundamental diagnostic for clinical practice and genetic counseling. These findings underscore the need to adapt long-read sequencing clinical workflows to fully characterize large repeat expansions at the nucleotide level.\u003c/p\u003e","manuscriptTitle":"Novel ATXN10 repeat motif patterns in Peruvian families modify disease age at onset","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-02-12 11:19:17","doi":"10.21203/rs.3.rs-5989910/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"2e7f37f2-10dd-4c7d-a621-e2fd1a7c0b49","owner":[],"postedDate":"February 12th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[{"id":44049589,"name":"Medical Genetics"}],"tags":[],"updatedAt":"2025-02-12T11:19:17+00:00","versionOfRecord":[],"versionCreatedAt":"2025-02-12 11:19:17","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-5989910","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-5989910","identity":"rs-5989910","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}