{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2026,10,3]],"date-time":"2026-10-03T17:45:41Z","timestamp":1791049541237,"version":"4.1.0"},"reference-count":38,"publisher":"MDPI AG","issue":"7","license":[{"start":{"date-parts":[[2023,7,6]],"date-time":"2023-07-06T00:00:00Z","timestamp":1688601600000},"content-version":"vor","delay-in-days":0,"URL":"https:\/\/creativecommons.org\/licenses\/by\/4.0\/"}],"funder":[{"name":"South-Eastern Norway Regional Health Authority","award":["469177153"],"award-info":[{"award-number":["469177153"]}]},{"name":"Swedish Brain Foundation","award":["469177153"],"award-info":[{"award-number":["469177153"]}]},{"name":"German Research Foundation DFG VO 2138\/7-1","award":["469177153"],"award-info":[{"award-number":["469177153"]}]},{"name":"Aga Khan University, Pakistan","award":["469177153"],"award-info":[{"award-number":["469177153"]}]},{"name":"The Wellcome Trust","award":["469177153"],"award-info":[{"award-number":["469177153"]}]},{"name":"The MRC","award":["469177153"],"award-info":[{"award-number":["469177153"]}]},{"name":"The MSA Trust","award":["469177153"],"award-info":[{"award-number":["469177153"]}]},{"name":"The National Institute for Health Research University College London Hospitals Biomedical Research Centre","award":["469177153"],"award-info":[{"award-number":["469177153"]}]},{"name":"The Michael J Fox Foundation (MJFF)","award":["469177153"],"award-info":[{"award-number":["469177153"]}]},{"name":"BBSRC","award":["469177153"],"award-info":[{"award-number":["469177153"]}]},{"name":"The Fidelity Trust","award":["469177153"],"award-info":[{"award-number":["469177153"]}]},{"name":"Rosetrees Trust","award":["469177153"],"award-info":[{"award-number":["469177153"]}]},{"name":"Ataxia UK","award":["469177153"],"award-info":[{"award-number":["469177153"]}]},{"name":"Brain Research UK","award":["469177153"],"award-info":[{"award-number":["469177153"]}]},{"name":"Sparks GOSH Charity","award":["469177153"],"award-info":[{"award-number":["469177153"]}]},{"name":"Alzheimer\u2019s Research UK (ARUK)","award":["469177153"],"award-info":[{"award-number":["469177153"]}]},{"name":"CureDRPLA","award":["469177153"],"award-info":[{"award-number":["469177153"]}]}],"content-domain":{"domain":[],"crossmark-restriction":false},"short-container-title":["Genes"],"abstract":"<jats:p>Spinocerebellar disorders are a vast group of rare neurogenetic conditions, generally characterized by overlapping clinical symptoms including progressive cerebellar ataxia, spastic paraparesis, cognitive deficiencies, skeletal\/muscular and ocular abnormalities. The objective of the present study is to identify the underlying genetic causes of the rare spinocerebellar disorders in the Pakistani population. Herein, nine consanguineous families presenting different spinocerebellar phenotypes have been investigated using whole exome sequencing. Sanger sequencing was performed for segregation analysis in all the available individuals of each family. The molecular analysis of these families identified six novel pathogenic\/likely pathogenic variants; ZFYVE26: c.1093del, SACS: c.1201C&gt;T, BICD2: c.2156A&gt;T, ALS2: c.2171-3T&gt;G, ALS2: c.3145T&gt;A, and B4GALNT1: c.334_335dup, and three already reported pathogenic variants; FA2H: c.159_176del, APTX: c.689T&gt;G, and SETX: c.5308_5311del. The clinical features of all patients in each family are concurrent with the already reported cases. Hence, the current study expands the mutation spectrum of rare spinocerebellar disorders and implies the usefulness of next-generation sequencing in combination with clinical investigation for better diagnosis of these overlapping phenotypes.<\/jats:p>","DOI":"10.3390\/genes14071404","type":"journal-article","created":{"date-parts":[[2023,7,7]],"date-time":"2023-07-07T01:41:36Z","timestamp":1688694096000},"page":"1404","update-policy":"https:\/\/doi.org\/10.3390\/mdpi_crossmark_policy","source":"Crossref","is-referenced-by-count":11,"title":["Genetic Investigation of Consanguineous Pakistani Families Segregating Rare Spinocerebellar Disorders"],"prefix":"10.3390","volume":"14","author":[{"ORCID":"https:\/\/orcid.org\/0000-0001-5962-2101","authenticated-orcid":false,"given":"Saadia Maryam","family":"Saadi","sequence":"first","affiliation":[{"name":"Human Molecular Genetics Laboratory, Health Biotechnology Division, National Institute for Biotechnology and Genetic Engineering College (NIBGE-C), Pakistan Institute of Engineering and Applied Sciences (PIEAS), Islamabad 44000, Pakistan"},{"name":"Department of Neuromuscular Disorders, UCL Queen Square Institute of Neurology, London WC1N 3BG, UK"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0001-9826-4490","authenticated-orcid":false,"given":"Elisa","family":"Cali","sequence":"additional","affiliation":[{"name":"Department of Neuromuscular Disorders, UCL Queen Square Institute of Neurology, London WC1N 3BG, UK"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Lubaba Bintee","family":"Khalid","sequence":"additional","affiliation":[{"name":"Department of Biological and Biomedical Sciences, Aga Khan University, Karachi 74000, Pakistan"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0009-0006-1538-2883","authenticated-orcid":false,"given":"Hammad","family":"Yousaf","sequence":"additional","affiliation":[{"name":"Human Molecular Genetics Laboratory, Health Biotechnology Division, National Institute for Biotechnology and Genetic Engineering College (NIBGE-C), Pakistan Institute of Engineering and Applied Sciences (PIEAS), Islamabad 44000, Pakistan"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Ghazala","family":"Zafar","sequence":"additional","affiliation":[{"name":"Department of Biological and Biomedical Sciences, Aga Khan University, Karachi 74000, Pakistan"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-3289-3128","authenticated-orcid":false,"given":"Haq Nawaz","family":"Khan","sequence":"additional","affiliation":[{"name":"Department of Biological and Biomedical Sciences, Aga Khan University, Karachi 74000, Pakistan"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Muhammad","family":"Sher","sequence":"additional","affiliation":[{"name":"Department of Allied Health Sciences, Iqra National University Swat Campus, Swat 19200, Pakistan"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-6719-3447","authenticated-orcid":false,"given":"Barbara","family":"Vona","sequence":"additional","affiliation":[{"name":"Institute of Human Genetics, University Medical Center G\u00f6ttingen, 37073 G\u00f6ttingen, Germany"},{"name":"Institute for Auditory Neuroscience and InnerEarLab, University Medical Center G\u00f6ttingen, 37075 G\u00f6ttingen, Germany"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-7168-8266","authenticated-orcid":false,"given":"Uzma","family":"Abdullah","sequence":"additional","affiliation":[{"name":"University Institute of Biochemistry and Biotechnology (UIBB), Pir Mehr Ali Shah Arid Agriculture University Rawalpindi (PMAS-AAUR), Rawalpindi 46300, Pakistan"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Naveed Altaf","family":"Malik","sequence":"additional","affiliation":[{"name":"Human Molecular Genetics Laboratory, Health Biotechnology Division, National Institute for Biotechnology and Genetic Engineering College (NIBGE-C), Pakistan Institute of Engineering and Applied Sciences (PIEAS), Islamabad 44000, Pakistan"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Joakim","family":"Klar","sequence":"additional","affiliation":[{"name":"Department of Immunology, Genetics and Pathology, Uppsala University and Science for Life Laboratory, P.O. Box 815, 751 08 Uppsala, Sweden"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0003-4900-9877","authenticated-orcid":false,"given":"Stephanie","family":"Efthymiou","sequence":"additional","affiliation":[{"name":"Department of Neuromuscular Disorders, UCL Queen Square Institute of Neurology, London WC1N 3BG, UK"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Niklas","family":"Dahl","sequence":"additional","affiliation":[{"name":"Department of Immunology, Genetics and Pathology, Uppsala University and Science for Life Laboratory, P.O. Box 815, 751 08 Uppsala, Sweden"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Henry","family":"Houlden","sequence":"additional","affiliation":[{"name":"Department of Neuromuscular Disorders, UCL Queen Square Institute of Neurology, London WC1N 3BG, UK"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-6723-6865","authenticated-orcid":false,"given":"Mathias","family":"Toft","sequence":"additional","affiliation":[{"name":"Institute of Clinical Medicine, University of Oslo, P.O. Box 1171, N-0318 Oslo, Norway"},{"name":"Department of Neurology, Oslo University Hospital, P.O. Box 4950 Nydalen, N-0424 Oslo, Norway"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-0683-5872","authenticated-orcid":false,"given":"Shahid Mahmood","family":"Baig","sequence":"additional","affiliation":[{"name":"Human Molecular Genetics Laboratory, Health Biotechnology Division, National Institute for Biotechnology and Genetic Engineering College (NIBGE-C), Pakistan Institute of Engineering and Applied Sciences (PIEAS), Islamabad 44000, Pakistan"},{"name":"Department of Biological and Biomedical Sciences, Aga Khan University, Karachi 74000, Pakistan"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0003-0567-5958","authenticated-orcid":false,"given":"Ambrin","family":"Fatima","sequence":"additional","affiliation":[{"name":"Department of Biological and Biomedical Sciences, Aga Khan University, Karachi 74000, Pakistan"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-6387-8019","authenticated-orcid":false,"given":"Zafar","family":"Iqbal","sequence":"additional","affiliation":[{"name":"Department of Neurology, Oslo University Hospital, P.O. Box 4950 Nydalen, N-0424 Oslo, Norway"}],"role":[{"vocabulary":"crossref","role":"author"}]}],"member":"1968","published-online":{"date-parts":[[2023,7,6]]},"reference":[{"key":"ref_1","doi-asserted-by":"crossref","first-page":"261","DOI":"10.1016\/B978-0-444-63916-5.00017-3","article-title":"Cerebellar Ataxia","volume":"Volume 159","author":"Marsden","year":"2018","journal-title":"Handbook of Clinical Neurology"},{"key":"ref_2","doi-asserted-by":"crossref","first-page":"154","DOI":"10.1016\/j.gaitpost.2017.11.024","article-title":"A systematic review of the gait characteristics associated with Cerebellar Ataxia","volume":"60","author":"Buckley","year":"2018","journal-title":"Gait Posture"},{"key":"ref_3","doi-asserted-by":"crossref","first-page":"1127","DOI":"10.1016\/S1474-4422(08)70258-8","article-title":"Hereditary spastic paraplegia: Clinical features and pathogenetic mechanisms","volume":"7","author":"Salinas","year":"2008","journal-title":"Lancet Neurol."},{"key":"ref_4","doi-asserted-by":"crossref","first-page":"257","DOI":"10.1159\/000206850","article-title":"Cognitive impairment in spinocerebellar degeneration","volume":"61","author":"Kawai","year":"2009","journal-title":"Eur. Neurol."},{"key":"ref_5","doi-asserted-by":"crossref","first-page":"638730","DOI":"10.3389\/fgene.2021.638730","article-title":"The history of gene hunting in hereditary spinocerebellar degeneration: Lessons from the past and future perspectives","volume":"12","author":"Yahia","year":"2021","journal-title":"Front. Genet."},{"key":"ref_6","doi-asserted-by":"crossref","first-page":"18","DOI":"10.1007\/s11910-019-0930-2","article-title":"Update on the genetics of spastic paraplegias","volume":"19","author":"Boutry","year":"2019","journal-title":"Curr. Neurol. Neurosci. Rep."},{"key":"ref_7","doi-asserted-by":"crossref","first-page":"462","DOI":"10.1097\/WCO.0000000000000585","article-title":"Hereditary ataxias and paraparesias: Clinical and genetic update","volume":"31","author":"Parodi","year":"2018","journal-title":"Curr. Opin. Neurol."},{"key":"ref_8","doi-asserted-by":"crossref","first-page":"150","DOI":"10.1097\/WCO.0000000000000774","article-title":"Cerebellar ataxias: An update","volume":"33","author":"Manto","year":"2020","journal-title":"Curr. Opin. Neurol."},{"key":"ref_9","doi-asserted-by":"crossref","first-page":"332","DOI":"10.1002\/mds.26944","article-title":"Overcoming the divide between ataxias and spastic paraplegias: Shared phenotypes, genes, and pathways","volume":"32","author":"Synofzik","year":"2017","journal-title":"Mov. Disord."},{"key":"ref_10","doi-asserted-by":"crossref","first-page":"165","DOI":"10.1038\/s41431-019-0508-0","article-title":"Estimating cumulative point prevalence of rare diseases: Analysis of the Orphanet database","volume":"28","author":"Lambert","year":"2020","journal-title":"Eur. J. Hum. Genet."},{"key":"ref_11","doi-asserted-by":"crossref","first-page":"174","DOI":"10.1159\/000358801","article-title":"The global epidemiology of hereditary ataxia and spastic paraplegia: A systematic review of prevalence studies","volume":"42","author":"Ruano","year":"2014","journal-title":"Neuroepidemiology"},{"key":"ref_12","doi-asserted-by":"crossref","unstructured":"Yousaf, H., Fatima, A., Ali, Z., Baig, S.M., Toft, M., and Iqbal, Z. (2022). A Novel Nonsense Variant in GRM1 Causes Autosomal Recessive Spinocerebellar Ataxia 13 in a Consanguineous Pakistani Family. Genes, 13.","DOI":"10.3390\/genes13091667"},{"key":"ref_13","doi-asserted-by":"crossref","first-page":"2948","DOI":"10.1093\/brain\/awz248","article-title":"Biallelic mutations in neurofascin cause neurodevelopmental impairment and peripheral demyelination","volume":"142","author":"Efthymiou","year":"2019","journal-title":"Brain"},{"key":"ref_14","doi-asserted-by":"crossref","first-page":"848","DOI":"10.1038\/ejhg.2017.54","article-title":"A missense variant in ITPR1 provides evidence for autosomal recessive SCA29 with asymptomatic cerebellar hypoplasia in carriers","volume":"25","author":"Klar","year":"2017","journal-title":"Eur. J. Hum. Genet."},{"key":"ref_15","first-page":"1","article-title":"SpliceAI-visual: A free online tool to improve SpliceAI splicing variant interpretation","volume":"17","author":"Filser","year":"2023","journal-title":"Human Genom."},{"key":"ref_16","doi-asserted-by":"crossref","first-page":"518","DOI":"10.1038\/s41467-020-20584-4","article-title":"AutoMap is a high performance homozygosity mapping tool using next-generation sequencing data","volume":"12","author":"Quinodoz","year":"2021","journal-title":"Nat. Commun."},{"key":"ref_17","doi-asserted-by":"crossref","first-page":"713","DOI":"10.1038\/nature05164","article-title":"The neurodegenerative disease protein aprataxin resolves abortive DNA ligation intermediates","volume":"443","author":"Ahel","year":"2006","journal-title":"Nature"},{"key":"ref_18","doi-asserted-by":"crossref","first-page":"1601","DOI":"10.1007\/s00438-022-01945-8","article-title":"NGS-driven molecular diagnosis of heterogeneous hereditary neurological disorders reveals novel and known variants in disease-causing genes","volume":"297","author":"Khan","year":"2022","journal-title":"Mol. Genet. Genom."},{"key":"ref_19","doi-asserted-by":"crossref","unstructured":"Nicolaou, P., Georghiou, A., Votsi, C., Middleton, L.T., Zamba-Papanicolaou, E., and Christodoulou, K. (2008). A novel c. 5308_5311delGAGA mutation in Senataxin in a Cypriot family with an autosomal recessive cerebellar ataxia. BMC Med. Genet., 9.","DOI":"10.1186\/1471-2350-9-28"},{"key":"ref_20","doi-asserted-by":"crossref","first-page":"275","DOI":"10.5582\/irdr.2018.01107","article-title":"Ataxia with ocular apraxia type 2 not responding to 4-aminopyridine: A rare mutation in the SETX gene in a Saudi patient","volume":"7","author":"Algahtani","year":"2018","journal-title":"Intractable Rare Dis. Res."},{"key":"ref_21","doi-asserted-by":"crossref","first-page":"49","DOI":"10.1007\/s10916-019-1519-7","article-title":"Prevalence and diagnosis of neurological disorders using different deep learning techniques: A meta-analysis","volume":"44","author":"Gautam","year":"2020","journal-title":"J. Med. Syst."},{"key":"ref_22","doi-asserted-by":"crossref","first-page":"841","DOI":"10.1097\/GIM.0b013e318217477f","article-title":"Consanguineous marriages, pearls and perils: Geneva International Consanguinity Workshop Report","volume":"3","author":"Hamamy","year":"2011","journal-title":"Genet. Med."},{"key":"ref_23","doi-asserted-by":"crossref","first-page":"3119","DOI":"10.1093\/brain\/awt227","article-title":"Defective autophagy in spastizin mutated patients with hereditary spastic paraparesis type 15","volume":"136","author":"Vantaggiato","year":"2013","journal-title":"Brain"},{"key":"ref_24","doi-asserted-by":"crossref","unstructured":"Shimazaki, H. (2017). Lysosomal Dysfunctions in Hereditary Spastic Paraplegias, in Lysosomes-Associated Diseases and Methods to Study Their Function. IntechOpen.","DOI":"10.5772\/intechopen.70142"},{"key":"ref_25","doi-asserted-by":"crossref","first-page":"116669","DOI":"10.1016\/j.jns.2020.116669","article-title":"Rare novel CYP2U1 and ZFYVE26 variants identified in two Pakistani families with spastic paraplegia","volume":"411","author":"Bibi","year":"2020","journal-title":"J. Neurol. Sci."},{"key":"ref_26","doi-asserted-by":"crossref","first-page":"500","DOI":"10.1111\/cge.12312","article-title":"Novel B4GALNT1 mutations in a complicated form of hereditary spastic paraplegia","volume":"86","author":"Wakil","year":"2014","journal-title":"Clin. Genet."},{"key":"ref_27","doi-asserted-by":"crossref","first-page":"118","DOI":"10.1016\/j.ajhg.2013.05.006","article-title":"Alteration of ganglioside biosynthesis responsible for complex hereditary spastic paraplegia","volume":"93","author":"Boukhris","year":"2013","journal-title":"Am. J. Hum. Genet."},{"key":"ref_28","doi-asserted-by":"crossref","first-page":"3618","DOI":"10.1093\/brain\/awt270","article-title":"Mutations in B4GALNT1 (GM2 synthase) underlie a new disorder of ganglioside biosynthesis","volume":"136","author":"Harlalka","year":"2013","journal-title":"Brain"},{"key":"ref_29","doi-asserted-by":"crossref","first-page":"1225","DOI":"10.1038\/ng1460","article-title":"Infantile-onset symptomatic epilepsy syndrome caused by a homozygous loss-of-function mutation of GM3 synthase","volume":"36","author":"Simpson","year":"2004","journal-title":"Nat. Genet."},{"key":"ref_30","doi-asserted-by":"crossref","first-page":"225","DOI":"10.1038\/ng1303","article-title":"Senataxin, the ortholog of a yeast RNA helicase, is mutant in ataxia-ocular apraxia 2","volume":"36","author":"Moreira","year":"2004","journal-title":"Nat. Genet."},{"key":"ref_31","doi-asserted-by":"crossref","unstructured":"Splinter, D., Tanenbaum, M.E., Lindqvist, A., Jaarsma, D., Flotho, A., Yu, K.L., Grigoriev, I., Engelsma, D., Haasdijk, E.D., and Keijzer, N. (2010). Bicaudal D2, dynein, and kinesin-1 associate with nuclear pore complexes and regulate centrosome and nuclear positioning during mitotic entry. PLoS Biol., 8.","DOI":"10.1371\/journal.pbio.1000350"},{"key":"ref_32","doi-asserted-by":"crossref","first-page":"946","DOI":"10.1016\/j.ajhg.2013.04.011","article-title":"Mutations in BICD2, which encodes a golgin and important motor adaptor, cause congenital autosomal-dominant spinal muscular atrophy","volume":"92","author":"Neveling","year":"2013","journal-title":"Am. J. Hum. Genet."},{"key":"ref_33","doi-asserted-by":"crossref","first-page":"3439","DOI":"10.1093\/nar\/24.17.3439","article-title":"Splice site prediction in Arabidopsis thaliana pre-mRNA by combining local and global sequence information","volume":"24","author":"Hebsgaard","year":"1996","journal-title":"Nucleic Acids Res."},{"key":"ref_34","doi-asserted-by":"crossref","first-page":"49","DOI":"10.1016\/0022-2836(91)90380-O","article-title":"Prediction of human mRNA donor and acceptor sites from the DNA sequence","volume":"220","author":"Brunak","year":"1991","journal-title":"J. Mol. Biol."},{"key":"ref_35","doi-asserted-by":"crossref","first-page":"74","DOI":"10.1016\/j.neuint.2007.04.010","article-title":"Molecular and cellular function of ALS2\/alsin: Implication of membrane dynamics in neuronal development and degeneration","volume":"51","author":"Hadano","year":"2007","journal-title":"Neurochem. Int."},{"key":"ref_36","doi-asserted-by":"crossref","first-page":"189","DOI":"10.1038\/ng1001-189","article-title":"The gene mutated in ataxia-ocular apraxia 1 encodes the new HIT\/Zn-finger protein aprataxin","volume":"29","author":"Moreira","year":"2001","journal-title":"Nat. Genet."},{"key":"ref_37","doi-asserted-by":"crossref","first-page":"184","DOI":"10.1038\/ng1001-184","article-title":"Early-onset ataxia with ocular motor apraxia and hypoalbuminemia is caused by mutations in a new HIT superfamily gene","volume":"29","author":"Date","year":"2001","journal-title":"Nat. Genet."},{"key":"ref_38","doi-asserted-by":"crossref","first-page":"1009","DOI":"10.1002\/glia.21172","article-title":"Central nervous system dysfunction in a mouse model of FA2H deficiency","volume":"59","author":"Potter","year":"2011","journal-title":"Glia"}],"container-title":["Genes"],"original-title":[],"language":"en","link":[{"URL":"https:\/\/www.mdpi.com\/2073-4425\/14\/7\/1404\/pdf","content-type":"unspecified","content-version":"vor","intended-application":"similarity-checking"}],"deposited":{"date-parts":[[2025,10,10]],"date-time":"2025-10-10T20:07:02Z","timestamp":1760126822000},"score":1,"resource":{"primary":{"URL":"https:\/\/www.mdpi.com\/2073-4425\/14\/7\/1404"}},"subtitle":[],"short-title":[],"issued":{"date-parts":[[2023,7,6]]},"references-count":38,"journal-issue":{"issue":"7","published-online":{"date-parts":[[2023,7]]}},"alternative-id":["genes14071404"],"URL":"https:\/\/doi.org\/10.3390\/genes14071404","relation":{},"ISSN":["2073-4425"],"issn-type":[{"value":"2073-4425","type":"electronic"}],"subject":[],"published":{"date-parts":[[2023,7,6]]}}}