Missense variants inTUBA4Acause myo-tubulinopathies

Missense variants in TUBA4A cause myo-tubulinopathies | medRxiv /* */ /* */ <!-- <!-- /*! * yepnope1.5.4 * (c) WTFPL, GPLv2 */ (function(a,b,c){function d(a){return"[object Function]"==o.call(a)}function e(a){return"string"==typeof a}function f(){}function g(a){return!a||"loaded"==a||"complete"==a||"uninitialized"==a}function h(){var a=p.shift();q=1,a?a.t?m(function(){("c"==a.t?B.injectCss:B.injectJs)(a.s,0,a.a,a.x,a.e,1)},0):(a(),h()):q=0}function i(a,c,d,e,f,i,j){function k(b){if(!o&&g(l.readyState)&&(u.r=o=1,!q&&h(),l.onload=l.onreadystatechange=null,b)){"img"!=a&&m(function(){t.removeChild(l)},50);for(var d in y[c])y[c].hasOwnProperty(d)&&y[c][d].onload()}}var j=j||B.errorTimeout,l=b.createElement(a),o=0,r=0,u={t:d,s:c,e:f,a:i,x:j};1===y[c]&&(r=1,y[c]=[]),"object"==a?l.data=c:(l.src=c,l.type=a),l.width=l.height="0",l.onerror=l.onload=l.onreadystatechange=function(){k.call(this,r)},p.splice(e,0,u),"img"!=a&&(r||2===y[c]?(t.insertBefore(l,s?null:n),m(k,j)):y[c].push(l))}function j(a,b,c,d,f){return q=0,b=b||"j",e(a)?i("c"==b?v:u,a,b,this.i++,c,d,f):(p.splice(this.i++,0,a),1==p.length&&h()),this}function k(){var a=B;return a.loader={load:j,i:0},a}var l=b.documentElement,m=a.setTimeout,n=b.getElementsByTagName("script")[0],o={}.toString,p=[],q=0,r="MozAppearance"in l.style,s=r&&!!b.createRange().compareNode,t=s?l:n.parentNode,l=a.opera&&"[object Opera]"==o.call(a.opera),l=!!b.attachEvent&&!l,u=r?"object":l?"script":"img",v=l?"script":u,w=Array.isArray||function(a){return"[object Array]"==o.call(a)},x=[],y={},z={timeout:function(a,b){return b.length&&(a.timeout=b[0]),a}},A,B;B=function(a){function b(a){var a=a.split("!"),b=x.length,c=a.pop(),d=a.length,c={url:c,origUrl:c,prefixes:a},e,f,g;for(f=0;f<d;f++)g=a[f].split("="),(e=z[g.shift()])&&(c=e(c,g));for(f=0;f<b;f++)c=x[f](c);return c}function g(a,e,f,g,h){var i=b(a),j=i.autoCallback;i.url.split(".").pop().split("?").shift(),i.bypass||(e&&(e=d(e)?e:e[a]||e[g]||e[a.split("/").pop().split("?")[0]]),i.instead?i.instead(a,e,f,g,h):(y[i.url]?i.noexec=!0:y[i.url]=1,f.load(i.url,i.forceCSS||!i.forceJS&&"css"==i.url.split(".").pop().split("?").shift()?"c":c,i.noexec,i.attrs,i.timeout),(d(e)||d(j))&&f.load(function(){k(),e&&e(i.origUrl,h,g),j&&j(i.origUrl,h,g),y[i.url]=2})))}function h(a,b){function c(a,c){if(a){if(e(a))c||(j=function(){var a=[].slice.call(arguments);k.apply(this,a),l()}),g(a,j,b,0,h);else if(Object(a)===a)for(n in m=function(){var b=0,c;for(c in a)a.hasOwnProperty(c)&&b++;return b}(),a)a.hasOwnProperty(n)&&(!c&&!--m&&(d(j)?j=function(){var a=[].slice.call(arguments);k.apply(this,a),l()}:j[n]=function(a){return function(){var b=[].slice.call(arguments);a&&a.apply(this,b),l()}}(k[n])),g(a[n],j,b,n,h))}else!c&&l()}var h=!!a.test,i=a.load||a.both,j=a.callback||f,k=j,l=a.complete||f,m,n;c(h?a.yep:a.nope,!!i),i&&c(i)}var i,j,l=this.yepnope.loader;if(e(a))g(a,0,l,0);else if(w(a))for(i=0;i (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];var j=d.createElement(s);var dl=l!='dataLayer'?'&l='+l:'';j.src='//www.googletagmanager.com/gtm.js?id='+i+dl;j.type='text/javascript';j.async=true;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-P4HH5NV'); Skip to main content Home About Submit ALERTS / RSS Search for this keyword Advanced Search Missense variants in TUBA4A cause myo-tubulinopathies View ORCID Profile Mridul Johari , View ORCID Profile Chiara Folland , View ORCID Profile Yoshihiko Saito , View ORCID Profile Machteld M. Oud , View ORCID Profile Jevin M. Parmar , View ORCID Profile Ana Töpf , View ORCID Profile Sergei Kurbatov , View ORCID Profile Maria Ampleeva , Ekaterina Y. Zakharova , View ORCID Profile Irina A. Chekmareva , Ksenia S. Shirokova , View ORCID Profile Dmitrii Atiakshin , Thatjana Gardeitchik , View ORCID Profile Erik-Jan Kamsteeg , View ORCID Profile Evita Medici , View ORCID Profile Laura Donker Kaat , View ORCID Profile Christine C. Bruels , View ORCID Profile Seth A. Stafki , View ORCID Profile Elicia A. Estrella , View ORCID Profile Hannah R. Littel , View ORCID Profile Louis M. Kunkel , View ORCID Profile Peter B. Kang , Ikeoluwa Osei-Owusu , Lynn Pais , Melaine O’Leary , Christina Austin-Tse , Anne O’Donnell-Luria , Brian Mangilog , View ORCID Profile Francesca Clementina Radio , View ORCID Profile Adele D’Amico , View ORCID Profile Andrea Ciolfi , View ORCID Profile Marco Tartaglia , Aurélien Perrin , Charles Van Goethem , Guilhem Sole , Marie-Laure Martin-Négrier , Mireille Cossée , View ORCID Profile Casie A. Genetti , View ORCID Profile Zaheer M. Valivullah , Vedrana Milic , View ORCID Profile Gordana Kovacevic , Ana Kosac , Cristiane A.M. Moreno , Clara Gontijo Camelo , Edmar Zanoteli , Michael C. Fahey , View ORCID Profile Alan H. Beggs , View ORCID Profile John Vissing , Volker Straub , View ORCID Profile Marco Savarese , View ORCID Profile Giorgio Tasca , Nicol Voermans , Nigel G. Laing , Bjarne Udd , View ORCID Profile Ichizo Nishino , View ORCID Profile Gianina Ravenscroft doi: https://doi.org/10.1101/2025.06.26.25330266 Mridul Johari 1 Harry Perkins Institute of Medical Research, Centre for Medical Research, University of Western Australia , Nedlands, WA, Australia Find this author on Google Scholar Find this author on PubMed Search for this author on this site ORCID record for Mridul Johari For correspondence: mridul.johari{at}uwa.edu.au gina.ravenscroft{at}uwa.edu.au Chiara Folland 1 Harry Perkins Institute of Medical Research, Centre for Medical Research, University of Western Australia , Nedlands, WA, Australia Find this author on Google Scholar Find this author on PubMed Search for this author on this site ORCID record for Chiara Folland Yoshihiko Saito 2 Department of Neuromuscular Research, National Institute of Neuroscience, National Center of Neurology and Psychiatry , Kodaira, Tokyo, Japan Find this author on Google Scholar Find this author on PubMed Search for this author on this site ORCID record for Yoshihiko Saito Machteld M. Oud 3 Department of Human Genetics, Donders Institute for Brain , Cognition and Behaviour, Radboud University Medical Center , Nijmegen, The Netherlands Find this author on Google Scholar Find this author on PubMed Search for this author on this site ORCID record for Machteld M. Oud Jevin M. Parmar 1 Harry Perkins Institute of Medical Research, Centre for Medical Research, University of Western Australia , Nedlands, WA, Australia Find this author on Google Scholar Find this author on PubMed Search for this author on this site ORCID record for Jevin M. Parmar Ana Töpf 4 John Walton Muscular Dystrophy Research Centre, NIHR Newcastle Biomedical Research Centre, Newcastle University and Newcastle Hospitals NHS Foundation Trust , Newcastle upon Tyne, United Kingdom Find this author on Google Scholar Find this author on PubMed Search for this author on this site ORCID record for Ana Töpf Sergei Kurbatov 5 Research Institute of Experimental Biology and Medicine, Voronezh State Medical University named after N.N. Burdenko , Voronezh, Russian Federation Find this author on Google Scholar Find this author on PubMed Search for this author on this site ORCID record for Sergei Kurbatov Maria Ampleeva 6 Independent Clinical Bioinformatics Laboratory , Moscow, Russia Find this author on Google Scholar Find this author on PubMed Search for this author on this site ORCID record for Maria Ampleeva Ekaterina Y. Zakharova 7 Research Centre for Medical Genetics , Moscow, Russia Find this author on Google Scholar Find this author on PubMed Search for this author on this site Irina A. Chekmareva 8 Federal State Budgetary Institution “National Medical Research Center of Surgery named after A. Vishnevsky”, Ministry of Health of the Russian Federation , Moscow, Russia Find this author on Google Scholar Find this author on PubMed Search for this author on this site ORCID record for Irina A. Chekmareva Ksenia S. Shirokova 5 Research Institute of Experimental Biology and Medicine, Voronezh State Medical University named after N.N. Burdenko , Voronezh, Russian Federation Find this author on Google Scholar Find this author on PubMed Search for this author on this site Dmitrii Atiakshin 9 RUDN University , Moscow, Russian Federation Find this author on Google Scholar Find this author on PubMed Search for this author on this site ORCID record for Dmitrii Atiakshin Thatjana Gardeitchik 10 Department of Human Genetics, Radboud University Medical Center , Nijmegen 6525 GA, the Netherlands Find this author on Google Scholar Find this author on PubMed Search for this author on this site Erik-Jan Kamsteeg 11 Dept Human Genetics , Radboudumc, Nijmegen, The Netherlands Find this author on Google Scholar Find this author on PubMed Search for this author on this site ORCID record for Erik-Jan Kamsteeg Evita Medici 12 Dept Neurology, Erasmus University Medical Center Rotterdam , The Netherlands Find this author on Google Scholar Find this author on PubMed Search for this author on this site ORCID record for Evita Medici Laura Donker Kaat 13 Dept Clinical Genetics, Erasmus University Medical Center Rotterdam , The Netherlands Find this author on Google Scholar Find this author on PubMed Search for this author on this site ORCID record for Laura Donker Kaat Christine C. Bruels 14 Greg Marzolf Jr. Muscular Dystrophy Center, Department of Neurology, University of Minnesota Medical School Find this author on Google Scholar Find this author on PubMed Search for this author on this site ORCID record for Christine C. Bruels Seth A. Stafki 14 Greg Marzolf Jr. Muscular Dystrophy Center, Department of Neurology, University of Minnesota Medical School Find this author on Google Scholar Find this author on PubMed Search for this author on this site ORCID record for Seth A. Stafki Elicia A. Estrella 15 Division of Genetics & Genomics, Boston Children’s Hospital and Harvard Medical School Find this author on Google Scholar Find this author on PubMed Search for this author on this site ORCID record for Elicia A. Estrella Hannah R. Littel 14 Greg Marzolf Jr. Muscular Dystrophy Center, Department of Neurology, University of Minnesota Medical School Find this author on Google Scholar Find this author on PubMed Search for this author on this site ORCID record for Hannah R. Littel Louis M. Kunkel 15 Division of Genetics & Genomics, Boston Children’s Hospital and Harvard Medical School Find this author on Google Scholar Find this author on PubMed Search for this author on this site ORCID record for Louis M. Kunkel Peter B. Kang 14 Greg Marzolf Jr. Muscular Dystrophy Center, Department of Neurology, University of Minnesota Medical School Find this author on Google Scholar Find this author on PubMed Search for this author on this site ORCID record for Peter B. Kang Ikeoluwa Osei-Owusu 16 Program in Medical and Population Genetics, Broad Institute of MIT and Harvard , Cambridge, MA, USA Find this author on Google Scholar Find this author on PubMed Search for this author on this site Lynn Pais 16 Program in Medical and Population Genetics, Broad Institute of MIT and Harvard , Cambridge, MA, USA Find this author on Google Scholar Find this author on PubMed Search for this author on this site Melaine O’Leary 16 Program in Medical and Population Genetics, Broad Institute of MIT and Harvard , Cambridge, MA, USA Find this author on Google Scholar Find this author on PubMed Search for this author on this site Christina Austin-Tse 16 Program in Medical and Population Genetics, Broad Institute of MIT and Harvard , Cambridge, MA, USA Find this author on Google Scholar Find this author on PubMed Search for this author on this site Anne O’Donnell-Luria 16 Program in Medical and Population Genetics, Broad Institute of MIT and Harvard , Cambridge, MA, USA Find this author on Google Scholar Find this author on PubMed Search for this author on this site Brian Mangilog 16 Program in Medical and Population Genetics, Broad Institute of MIT and Harvard , Cambridge, MA, USA Find this author on Google Scholar Find this author on PubMed Search for this author on this site Francesca Clementina Radio 17 Molecular Genetics and Functional Genomics, Bambino Gesù Children’s Hospital , IRCCS, 00146, Rome, Italy Find this author on Google Scholar Find this author on PubMed Search for this author on this site ORCID record for Francesca Clementina Radio Adele D’Amico 18 Neuromuscular and neurodegenerative disorders, Bambino Gesù Children’s Hospital , IRCCS, 00146, Rome, Italy Find this author on Google Scholar Find this author on PubMed Search for this author on this site ORCID record for Adele D’Amico Andrea Ciolfi 17 Molecular Genetics and Functional Genomics, Bambino Gesù Children’s Hospital , IRCCS, 00146, Rome, Italy Find this author on Google Scholar Find this author on PubMed Search for this author on this site ORCID record for Andrea Ciolfi Marco Tartaglia 17 Molecular Genetics and Functional Genomics, Bambino Gesù Children’s Hospital , IRCCS, 00146, Rome, Italy Find this author on Google Scholar Find this author on PubMed Search for this author on this site ORCID record for Marco Tartaglia Aurélien Perrin 19 Laboratoire de Génétique Moléculaire, Centre Hospitalier Universitaire de Montpellier , 34093 Montpellier, France Find this author on Google Scholar Find this author on PubMed Search for this author on this site Charles Van Goethem 19 Laboratoire de Génétique Moléculaire, Centre Hospitalier Universitaire de Montpellier , 34093 Montpellier, France Find this author on Google Scholar Find this author on PubMed Search for this author on this site Guilhem Sole 20 Neurology and Neuromuscular Diseases Department, Neuromuscular Reference Centre AOC , FILNEMUS, EURO-NMD, Pellegrin Hospital, Bordeaux University Hospitals , Bordeaux, France Find this author on Google Scholar Find this author on PubMed Search for this author on this site Marie-Laure Martin-Négrier 21 Pathology Department, University Hospital of Bordeaux , France. Univ. Bordeaux, CNRS UMR5293, Bordeaux, France Find this author on Google Scholar Find this author on PubMed Search for this author on this site Mireille Cossée 19 Laboratoire de Génétique Moléculaire, Centre Hospitalier Universitaire de Montpellier , 34093 Montpellier, France Find this author on Google Scholar Find this author on PubMed Search for this author on this site Casie A. Genetti 22 Division of Genetics and Genomics, The Manton Center for Orphan Disease Research,Boston Children’s Hospital, Harvard Medical School , Boston MA 02115, USA Find this author on Google Scholar Find this author on PubMed Search for this author on this site ORCID record for Casie A. Genetti Zaheer M. Valivullah 23 Center for Mendelian Genomics, Broad Institute Harvard , Cambridge, MA 02142, USA Find this author on Google Scholar Find this author on PubMed Search for this author on this site ORCID record for Zaheer M. Valivullah Vedrana Milic 24 Clinic for Neurology and Psychiatry for Children and Youth, Belgrade, Serbia Find this author on Google Scholar Find this author on PubMed Search for this author on this site Gordana Kovacevic 25 Mother and Child Health Care Institute, Belgrade, Serbia Find this author on Google Scholar Find this author on PubMed Search for this author on this site ORCID record for Gordana Kovacevic Ana Kosac 26 Clinic for Neurology and Psychiatry for Children and Youth , Belgrade, Serbia Find this author on Google Scholar Find this author on PubMed Search for this author on this site Cristiane A.M. Moreno 27 Department of Neurology, Faculdade de Medicina da Universidade de São Paulo (FMUSP) , São Paulo. Brazil Find this author on Google Scholar Find this author on PubMed Search for this author on this site Clara Gontijo Camelo 27 Department of Neurology, Faculdade de Medicina da Universidade de São Paulo (FMUSP) , São Paulo. Brazil Find this author on Google Scholar Find this author on PubMed Search for this author on this site Edmar Zanoteli 27 Department of Neurology, Faculdade de Medicina da Universidade de São Paulo (FMUSP) , São Paulo. Brazil Find this author on Google Scholar Find this author on PubMed Search for this author on this site Michael C. Fahey 28 Department of Paediatrics Monash University , Melbourne, VIC, Australia Find this author on Google Scholar Find this author on PubMed Search for this author on this site Alan H. Beggs 22 Division of Genetics and Genomics, The Manton Center for Orphan Disease Research,Boston Children’s Hospital, Harvard Medical School , Boston MA 02115, USA Find this author on Google Scholar Find this author on PubMed Search for this author on this site ORCID record for Alan H. Beggs John Vissing 29 Copenhagen Neuromuscular Center, Rigshospitalet, University of Copenhagen , Copenhagen, Denmark Find this author on Google Scholar Find this author on PubMed Search for this author on this site ORCID record for John Vissing Volker Straub 4 John Walton Muscular Dystrophy Research Centre, NIHR Newcastle Biomedical Research Centre, Newcastle University and Newcastle Hospitals NHS Foundation Trust , Newcastle upon Tyne, United Kingdom Find this author on Google Scholar Find this author on PubMed Search for this author on this site Marco Savarese 30 Folkhälsan Research Center , Helsinki, Finland Find this author on Google Scholar Find this author on PubMed Search for this author on this site ORCID record for Marco Savarese Giorgio Tasca 4 John Walton Muscular Dystrophy Research Centre, NIHR Newcastle Biomedical Research Centre, Newcastle University and Newcastle Hospitals NHS Foundation Trust , Newcastle upon Tyne, United Kingdom Find this author on Google Scholar Find this author on PubMed Search for this author on this site ORCID record for Giorgio Tasca Nicol Voermans 31 Department of Neurology, Donders Institute for Brain , Cognition and Behaviour, Amalia Children’s Hospital, Radboud University Medical Centre , Nijmegen, The Netherlands Find this author on Google Scholar Find this author on PubMed Search for this author on this site Nigel G. Laing 1 Harry Perkins Institute of Medical Research, Centre for Medical Research, University of Western Australia , Nedlands, WA, Australia Find this author on Google Scholar Find this author on PubMed Search for this author on this site Bjarne Udd 30 Folkhälsan Research Center , Helsinki, Finland Find this author on Google Scholar Find this author on PubMed Search for this author on this site Ichizo Nishino 2 Department of Neuromuscular Research, National Institute of Neuroscience, National Center of Neurology and Psychiatry , Kodaira, Tokyo, Japan Find this author on Google Scholar Find this author on PubMed Search for this author on this site ORCID record for Ichizo Nishino Gianina Ravenscroft 1 Harry Perkins Institute of Medical Research, Centre for Medical Research, University of Western Australia , Nedlands, WA, Australia Find this author on Google Scholar Find this author on PubMed Search for this author on this site ORCID record for Gianina Ravenscroft For correspondence: mridul.johari{at}uwa.edu.au gina.ravenscroft{at}uwa.edu.au Abstract Full Text Info/History Metrics Data/Code Preview PDF Abstract Tubulinopathies encompass a wide spectrum of disorders resulting from variants in genes encoding α- and β-tubulins, the key components of microtubules. While previous studies have linked de novo or dominantly inherited TUBA4A missense variants to neurodegenerative phenotypes, including amyotrophic lateral sclerosis, frontotemporal dementia, hereditary spastic ataxia, and more recently, an isolated report of congenital myopathy, the full phenotypic and genotypic spectrum of TUBA4A -related disorders remains incompletely characterised. In this multi-centre study, we identified 13 novel TUBA4A missense variants in 31 individuals from 19 unrelated families. Remarkably, affected individuals in 17 families presented with a primary axial myopathy without any identified CNS involvement or history of such disease. In the remaining two families, we observed probands with cerebellar ataxia and epilepsy accompanying proximal and axial muscle weakness, establishing the first documented association between TUBA4A variants and multisystem proteinopathy. Our cohort exhibited diverse genotypes and associated inheritance patterns: four families demonstrated autosomal dominant transmission through heterozygous variants in TUBA4A , three probands had homozygous TUBA4A variants, where the biallelic genotype was found to be associated with the disease, and the heterozygous carriers were asymptomatic; five probands carried de novo variants, and nine probands with heterozygous TUBA4A variants were classified as "isolated-sporadic cases" where parental samples were unavailable. Clinical phenotypes ranged from mild to severe myopathy, predominantly affecting the axial and paraspinal muscles. We observed a range of disease onset, from congenital to late adulthood. Creatine kinase levels were also variable, ranging from normal to highly elevated. Cardiac function remained preserved across the cohort. Muscle biopsies revealed a range of pathologies, including myofibre size variation, myofibre atrophy, nemaline bodies, core-like regions, internal nuclei, and endomysial fibrosis. Immunohistochemical staining showed evidence of proteinopathy, with autophagic features and TUBA4A accumulation in patient myofibres. Complementary in silico and in vitro investigations suggested that the identified TUBA4A substitutions cause significant protein abnormalities and may differentially impact microtubule dynamics. Our findings establish myo-tubulinopathies as distinct clinical entities, encompassing both primary myopathies and multisystem proteinopathies with muscle involvement. This study broadens the phenotypic and genotypic spectrum of TUBA4A -related disorders beyond autosomal dominant or de novo mechanisms and neurodegenerative presentations. These results underscore the importance of considering TUBA4A variants in the differential diagnosis of axial myopathies and multisystem proteinopathies, regardless of central nervous system (CNS) involvement. Introduction Tubulinopathies arise from defective microtubule function or assembly due to pathogenic variants in the genes encoding different tubulin isotypes. 1 The term ‘tubulinopathy’ was first used to describe diseases associated with brain malformations arising from defects in cortical development and faulty neuronal migration, which are at the end of the phenotypic spectrum. 1 However, ‘tubulinopathies’ are now an umbrella term for a wide spectrum of ciliary, zygotic arrest and early developmental failure, neurological and neurodegenerative phenotypes caused by pathogenic defects in microtubule units and associated proteins. Microtubules, a fundamental cytoskeletal component in all forms of life, exhibit dynamic instability—a continuous cycle of growth and shrinkage that is essential for various cellular functions, including maintaining cell shape, enabling intracellular transport, and participating in cell division. 2 – 4 Microtubules are composed of different tubulin subunits encoded by several genes, resulting in various tubulin isotypes. The most common of these, α- and β-tubulin, form a noncovalent heterodimer, which serves as the building blocks of microtubules. 5 , 6 In humans, nine genes encode different α-tubulin isotypes, each of which can potentially pair with any of the ten β-tubulin isotypes, contributing to microtubule diversity and functional specialisation. Amongst the nine α-tubulin isotypes, TUBA4A encodes tubulin alpha 4a. Smith and colleagues first observed TUBA4A -related microtubule disarray in patients with familial amyotrophic lateral sclerosis (ALS), demonstrating that TUBA4A mutants have an impaired ability to form α- and β-tubulin dimers, which disrupts microtubule dynamics and stability through a dominant-negative mechanism. 7 Later, pathogenic variants in TUBA4A were reported in patients with frontotemporal dementia (FTD), 8 , 9 Parkinson’s disease, 10 hereditary spastic ataxia, 11 , 12 and zygotic arrest. 13 , 14 Recently, a study identified a de novo variant in TUBA4A associated with an isolated congenital myopathy phenotype in two unrelated probands. 15 The pathogenic variants observed so far in TUBA4A are not restricted to any domain; how these variants result in a specific phenotype remains unknown. Impaired dimerisation and microtubule instability are shared functional defects of most mutant tubulin proteins, including TUBA4A mutants. 7 , 16 – 19 However, these gain-of-function pathomechanisms, primarily due to missense variants, can lead to vastly different phenotypes. 7 , 12 Additionally, heterozygous protein truncating variants in TUBA4A , which escape nonsense-mediated decay and result in loss of parts of C-terminal TUBA4A, ultimately forming a shorter protein, have also been associated with ALS and FTD phenotypes. 7 , 20 In our study, we present a gamut of TUBA4A -related primary myopathies and multisystem proteinopathies, providing detailed clinical and functional insights. We identified 13 variants in 31 affected individuals from 19 unrelated families. We observed autosomal dominant, including de novo , and recessive inheritance. Disease onset ranged from congenital to late adulthood, predominantly affecting axial and proximal muscles. Notably, unlike other tubulinopathies, 17 out of 19 families neither exhibited any central nervous system impairment nor had a history of such disease. In the remaining two cases, multisystem involvement of myopathy, cerebellar ataxia and epilepsy were seen. Through in silico , in vitro , histopathological and immunohistochemical analyses, we characterise the phenotypic and molecular spectrum of TUBA4A -related myopathies and multisystem proteinopathies – henceforth referred to as ‘myo-tubulinopathies’. Materials and Methods Patients and clinical examinations All probands and affected members underwent clinical neuromuscular and neurological examinations. The inclusion criteria for this cohort were a presumptive diagnosis of myopathy and the presence of a rare coding variant in TUBA4A, as identified through diagnostic or research testing. We collected blood and/or saliva samples for DNA extraction from affected and unaffected members from 19 families. Muscle imaging was obtained as part of the routine diagnostic work-up and was available for inclusion in this study for some patients, with consent. Electrophysiological examination results, including nerve conduction studies and needle electromyography (EMG), as well as serum creatine kinase (CK) measurements, were obtained for most patients. Aggregated clinical data was available for 21 patients. The studies were conducted per the Declaration of Helsinki, and ethical approval was obtained through institutional review boards (Supplementary Data 1(available upon request)). Muscle biopsy and immunohistochemical studies Muscle biopsies were obtained from a subset of affected patients ( n =13). Routine muscle histopathological studies were performed, including hematoxylin and eosin (H&E) staining, modified Gomori trichrome staining, and NADH tetrazolium reductase (NADH-TR) staining. 21 DAB immunostaining was performed using mouse monoclonal anti-myotilin (clone RSO34, 1:20, LEICA Biosystems Newcastle Ltd, UK) and mouse monoclonal anti-desmin (clone D33, 1:70, Richard-Allan Scientific, USA), with mouse ExtrAvidin Peroxidase Staining Kit (EXTRA2, Merck KGaA, Darmstadt, Germany). Microscopic images were obtained using a NIKON ECLIPSE Ci microscope equipped with an OLYMPUS ColorView II camera. Additional staining for aggregate pathology was performed on patient samples (Patients 5, 8, 9, 11, 12, and 24, n = 6 ). TUBA4A immunostaining was performed using a rabbit monoclonal anti-TUBA4A antibody (clone EPR13477(B), 1:100, Abcam, UK) after heat-mediated antigen retrieval with citrate buffer (pH 6.0) for 8 min at 91°C. SQSTM1 (p62) immunostaining used mouse monoclonal anti-SQSTM1 antibody (clone D-3, 1:200, Santa Cruz Biotechnology, Inc, USA). TARDBP (TDP-43) staining was performed using a rabbit polyclonal anti-TDP-43 antibody (1:1,000, Proteintech, Japan) after heat-mediated antigen retrieval with TE buffer (pH 9.0) for 8 minutes at 95°C. Electron microscopy and quantification analysis Ultrathin resin sections (70-80 nm) were prepared for electron microscopy from patients 8, 9, 25 and 26 and two healthy control individuals and examined using a FEI Tecnai Spirit Transmission Electron Microscope operating at 120 kV. Electron micrographs were obtained using the Gatan Digital Micrograph (Gatan, Inc, USA). For microtubule quantification, ultrathin resin sections from Pat.8 and Pat.9 were used. Ten microtubules were identified in these two patients and only five in controls. Average diameters were calculated, and an unpaired t-test was used for statistical calculations. DNA sequencing and analysis Targeted gene panel sequencing, short read (sr)-exome sequencing (ES) and sr-genome sequencing (GS) were carried out on probands and available family members (Supplementary Data 1(available upon request)). Sanger sequencing was performed to confirm the variants identified in the probands and co-segregate the variants with disease in available family members. Single nucleotide variant (SNV) and indel analyses were done through a) seqr- a web-based platform hosted by the Centre for Population Genomics (CPG; Garvan Institute of Medical Research, Murdoch Children’s Research Institute) and the Centre for Mendelian Genomics (CMG; the Broad Institute), 22 b) RD-Connect Genome Phenome Analysis Platform (GPAP), 23 and custom in-house pipelines using a minor allele frequency (MAF) σ; 0.0001 in the Genome Aggregation Database (gnomAD) v2.1.1 (hg19), 24 and v3.1.2 (hg38). 25 TUBA4A variant analysis and interpretation Variants in TUBA4A were annotated on NM_006000.3 (ENST00000248437.9, MANE Select) and NP_005991.1 (protein). Variant frequency reanalysis was performed using the updated gnomAD v4.1.0. Identified variants were assessed using various in silico tools (Supplementary Data 2) per current ACMG/AMP guidelines. The variant assessment was refined using the germline variant classifier VarSome, 26 with a modified pathogenicity interpretation based on the evidence levels available, including those collected during this study. In-silico modelling of TUBA4A variants Multiple sequence alignment To examine the conservation of substituted amino acid positions, we performed multiple sequence alignments of TUBA4A orthologs across different species and human TUBA4A paralogs. TUBA4A ortholog and paralog protein sequences were retrieved from UniProt using their respective accession codes. Alignments were performed using the default MAFFT algorithm in Jalview (v2.11.2). Calculation of free energy changes and TUBA4A aggregation prediction We used FoldX (v5.0) 27 to investigate the impact of TUBA4A variants on protein stability and to show the free energy changes between the wild-type and mutant proteins. Free energy change predictions were only performed on missense substitutions within high confidence regions (pLDDT > 90) of the AlphaFold2-predicted protein structure. 28 The AlphaFold2-derived wild- type TUBA4A protein structure, based on NM_006000.3, was energy-minimised and prepared for mutagenesis with the ‘RepairPDB’ and ‘BuildModel’ commands in FoldX, respectively. Subsequent free energy calculations were performed with the ‘Stability’ command, and differences in free energy were determined by subtracting the free energy value of the wild- type protein monomer from that of the mutant protein (ΔG MUT - ΔG WT ). Calculations were replicated 10 times with default parameters for each substitution. Substitutions were classified as having a destabilising effect on protein folding if the free energy difference (ΔΔG) was >1.6 kcal/mol. 27 , 29 , 30 Furthermore, we calculated the significance of FoldX stability predictions per- variant by comparing the per-variant values against a conservative destabilisation threshold of 1.6 kcal/mol using a bootstrapping approach. Analyses and plotting were performed in RStudio (v2024.12.1+563) R (v4.4.3). Aggrescan 4D was used to examine the aggregation propensity of wild-type and mutant TUBA4A. 31 , 32 Since there is no TUBA4A 3D protein structure derived from X-ray diffraction or solution NMR, Aggrescan utilises modelling approaches in PDB/mmCIF format to predict the aggregation propensities of TUBA4A. We used the A4D Dynamic mode, which integrates the CABS-flex tool to model protein flexibility and incorporate this information into the assessment of aggregation properties. 33 In this approach, the simulation begins with a FoldX- optimised input structure as the starting point. 27 Aggregation propensity scores were calculated for all 448 amino acids of TUBA4A across both wild-type and mutant proteins. The normalised average aggregation propensity score of each TUBA4A mutant protein was calculated relative to wild-type, using the Aggrescan4D-derived scores. Analyses and plotting were performed in RStudio (v2023.06.1) R (v4.3.3). TUBA4A structural modelling and in silico mutagenesis The predicted wild-type TUBA4A protein structure, based on NM_006000.3/NP_005991.1, was retrieved from the AlphaFold Protein Structure Database 34 using its UniProt accession code ( P68366 ). Three-dimensional protein structures were visualised using PyMol (v. 2.5.2). To examine the effects of variants on three-dimensional protein structure, in silico mutagenesis for identified missense substitutions was performed through the PyMol ‘mutagenesis’ function. To examine whether the variants affect any binding sites, an experimentally validated model of mouse TUBA4A-TUBB2A with kinesin (PDB: 8IXF) 35 was visualised, and images were exported using Mol*. 36 In vitro over-expression of TUBA4A variants in COS1 cells Wild-type human TUBA4A ORF was cloned into pcDNA3 to produce an N-terminally HA- tagged construct. The observed missense variants were introduced to the construct by site- directed mutagenesis. Wild-type and mutant TUBA4A were overexpressed in COS1 cells to study the localisation of TUBA4A and microtubule morphology. COS1 cells were cultured in Dulbecco’s Modified Eagle Medium (DMEM) supplemented with 10% fetal calf serum (FCS). Cells were grown on glass coverslips and, at approximately 60% confluence, transfected with Lipofectamine 2000 (Invitrogen) according to the manufacturer’s procedure. Cells were transfected with either WT or TUBA4A -myopathy variants: p.(Thr179Ile), p.(Leu227Phe), p.(Ser241Phe), p.(Glu254Ala), p.(Glu254Gln), p.(Glu254Lys), p.(Glu254Gly), p.(Gly354Asp). Additionally, we also transfected cells with previously identified TUBA4A ALS-causing substitution p.(Arg105Cys) and p.(Arg320Cys). 7 Upon 48 hours of incubation, the cells were fixed with ice-cold methanol for 10 minutes at room temperature (RT) and blocked with 2% bovine serum albumin (BSA) in PBS, followed by 1-hour incubation with the primary anti-HA antibody (mouse monoclonal, Sigma-Aldrich, Zwijndrecht, Netherlands), a PBS wash and another 1 hr incubation with the secondary anti-mouse Alexa Fluor-488 antibody (ThermoFisher Scientific Waltham, USA). The coverslips were embedded in Fluoromount-D with DAPI (Southern Biotech, Birmingham, AL, USA) on a microscopic glass slide. Then, the coverslips were analysed at 63x magnification with a Zeiss Axio Imager Z2 microscope (Zeiss, Sliedrecht, Netherlands) equipped with an ApoTome slider. The experiments were performed in triplicate. The statistical difference for the abnormal pattern was calculated for the wildtype compared to the TUBA4A-mutants using a two-way ANOVA followed by Tukey’s post hoc test. Data availability statement All relevant imaging and biological materials included in this study are available from the authors (or commercial providers). Sensitive sr-ES/sr-GS data of patients and family members are deposited in seqr and RD-Connect GPAP. Due to the extensive clinical details in Supplementary Data 1, which may contain potentially identifying information, this section has been removed from the preprint version. Readers interested in the detailed case-level data should contact the corresponding author for access, subject to appropriate data sharing agreements and ethical guidelines. Results Clinical features in myo-tubulinopathies A summary of clinical findings from 21 affected individuals is provided in Table 1 . Detailed clinical descriptions are provided in the (Supplementary Data 1(available upon request)). In this myo-tubulinopathy cohort, the mean age of onset was 15.1 years (range: 0 – 60 years). Four patients had onset of neuromuscular symptoms at birth. Two patients died during the first year of life (Pat.9 and Pat.10). Within the cohort, 56% ( n = 10/18) were non-ambulant. Most patients exhibited mild to moderate proximal and distal muscle weakness. Proximal upper and lower limb strength was more frequently affected than distal strength. While more than half of the cohort had normal to mildly reduced distal limb strength, a smaller subset showed moderate to severe weakness, particularly in the lower limbs ( Table 1 ). View this table: View inline View popup Download powerpoint Table 1: Summary of clinical features of affected individuals in our myo-tubulinopathy cohort with disease causing variants in TUBA4A. Each feature is shown as x/y under "Total", where ’x’ is the number of individuals exhibiting the feature, and y is the number of individuals for whom data were available in that category. The corresponding percentage is calculated from these values. Denominators vary due to missing data. A representation of muscle strength and weakness in our cohort, based on the MRC scale, is shown in Fig. 1A . An asymmetrical muscle weakness pattern was observed in 9% of patients. Axial muscle weakness was a consistent feature in our myo-tubulinopathy cohort (18/19). Nearly half of the patients also showed facial muscle involvement, and a smaller proportion (25%) presented with ptosis. Notably, ophthalmoplegia was identified in two individuals, and ophthalmoparesis in one — an uncommon finding in myopathies. Respiratory muscle weakness was observed in most cases (79%). Serum CK data was available for 21 individuals in the cohort, and three of these patients had normal CK. The mean level was 1,468.5 IU/l (range: 143-2,794 IU/l). For patients where muscle MRIs were available ( Fig. 1B ), fatty degeneration of gluteus minimus and erector spinae and often preserved gluteus maximus muscles. Accordingly, axial and paraspinal muscle weakness was observed as the most common feature. Download figure Open in new tab Fig. 1: TUBA4A -related myopathy presents with variable patterns of muscle weakness, most consistently affecting axial muscles. A) Spectrum of muscle weakness observed in patients with variants in TUBA4A . MuscleViz ( https://muscleviz.github.io ) was used to visualise the weakness using the MRC scale for muscle strength. Muscle weakness patterns and age of onset observed in our myo-tubulinopathy cohort are shown by the body diagrams. B) Axial T1-weighted MR images of muscles of the gluteal region, thighs, and calves from six patients with TUBA4A variants, arranged from left to right in order of increasing disease severity. Each panel includes the patient’s sex, age at imaging, and specific TUBA4A variant. In 17 out of 19 families, at the last assessment, no clinical signs or history of a neurodegenerative disease including hereditary spastic ataxia, ALS, dementia or Alzheimer disease were observed or stated. However, in two patients we observed multisystem involvement presenting as cerebellar ataxia and/or epileptic seizures along with a myofibrillar myopathy with prominent axial muscle weakness. Muscle pathology and protein accumulation pattern in myo-tubulinopathies Histopathological findings, where detailed data was available ( n = 13), are summarised separately in Table 2 , all muscle biopsies showed marked variation in myofibre size. Otherwise, there were no unifying histopathological hallmarks across the cohort. Eleven biopsies exhibited internally located nuclei, six showed predominance of type I myofibres and nine revealed endomysial fibrosis ( Fig. 2A ). Additionally, autophagic vacuoles and myofibrillar disorganisation were observed in four biopsies each. Download figure Open in new tab Fig. 2: TUBA4A -related myopathy shows a spectrum of histological, immunohistochemical and ultrastructural findings. A) (i-ix) Histochemical staining of patient muscle biopsies: i-iii) Hematoxylin & eosin (H&E) stain showing myofibre size variation, small angular myofibres, internal nucleation and endomysial fibrosis. iv-vi) Modified Gomori’s trichome staining showing presence of Nemaline bodies, spheroid or cytoplasmic bodies and occasional rimmed vacuoles. vii-ix) NADH-TR staining showing myofibre type and marked atrophy of type 1 myofibres along with minicores, large cores and moth eaten myofibres. x-xii) ATPase staining showing variability in myofibre size and atrophy, type 1 myofibre predominance and immature myofibres. B. (i-ix) DAB Immunohistochemical staining of patient and control muscle biopsies: i-iii) Increased immunoreactivity for p62/SQQSTM1 staining in all cases examined. iv-vi) However, TDP-43/TARDBP staining showed variable immunoreactivity for available muscle samples. vii-xii) Staining for anti-TUBA4A also showed variable immunoreactivity in different patients as compared to control muscle sample. C. (i-vi) Electron microscopy of patient muscles: i-ii) Ultrastructural analysis of Pat.8 muscle biopsy showing myofibrillar disarray and autophagic vacuoles. iii-iv) Pat.25 showed presence of Nemaline bodies while glycogen accumulation is seen in Pat.9. v-vi) Internal nucleation is seen in muscle biopsies of both Pat.8 and Pat.9. View this table: View inline View popup Download powerpoint Table 2: Histopathological features observed in individuals from our myo-tubulinopathy cohort. To further investigate accompanying proteinopathy features, immunohistochemical analysis was performed in a subset of patients. All examined cases ( n =4) demonstrated increased immunoreactivity for p62/SQSTM1 staining, indicating enhanced accumulation of this protein in myofibres. In contrast, TDP-43/TARDBP staining showed pronounced phosphorylated TDP-43 accumulations in some biopsies and not in others. Similarly, anti-TUBA4A staining revealed heterogenous levels of TUBA4A positive accumulations in patients’ biopsies when compared to control muscle samples, which may reflect differences in the underlying variants ( Fig. 2B ). Additionally, a subset of patients also exhibited peculiar ultrastructural features. Nemaline bodies were identified in four patients (Pat.9, Pat.12, Pat.25 and Pat.26, Fig. 2A -iv, Fig. 2C -iii). Other rare features included spheroid bodies, multiminicores, moth-eaten myofibres, abnormal glycogen accumulation, and small angular myofibres ( Fig. 2A , 2C). Identification of variants in TUBA4A Within the cohort we identified 13 different missense TUBA4A variants in 19 families. A representation of the observed variants in TUBA4A is depicted in Fig. 3A . Autosomal dominant ( n =4 families), de novo ( n =5 probands) and autosomal recessive ( n =3 probands) variants were observed. In nine sporadic cases with heterozygous variants the mode of inheritance could not be clarified due to lack of additional familial samples. At least in one family (Pat.14), the deceased parent of the proband was indicated as affected. Aggregated genetic findings are summarised in Table 3 . Interestingly, in four unrelated cases, four different heterozygous variants were identified which impacted the same amino-acid residue, Glu254, resulting in substitutions p.(Glu254Gly) and p.(Glu25Lys), confirmed to have arisen de novo in the probands, and p.(Glu254Ala) and p.(Glu254Gln) in two sporadic cases. Two of these variants, p.(Glu254Gln) and p.(Glu254Gly) were identified in the patients who died within the first year of life. Download figure Open in new tab Fig. 3: Modelling of variants in TUBA4A and in silico assessment of their impact suggests distinct pathomechanisms associated with different TUBA4A variants. A) Gene model of TUBA4A and 2D model of TUBA4A protein, showing the position of different myopathy variants (indicated as ‘coloured lollipops’) observed in our cohort. p.(Thr179Ile), p.(Leu227Phe) and p.(Glu284Lys) are in bold to indicate being observed in multiple families. p.(Ala12Thr), p.(Ser241Phe) and p.(Gly354Asp) are indicated with a ‘yellow lollipop’ to indicate homozygosity. Domain information for TUBA4A protein is obtained from Interpro-CATH gene3D. Exon 1 codes for the initiation site – methionine which forms the N-terminal domain. Thereafter, 2-268 amino acids form the GTPase domain, 269- 383 form the C-terminal domain and 384-448 form the Helix hairpin bin. All our observed variants are in the GTPase domain and the C-terminal domain. B) Conservation of Tubulins across species showed that all identified variants in our myopathy cohort are at highly conserved sites. C) Conservation of identified TUBA4A variant amino acid residues across other human tubulin paralogs. Conserved amino acids are highlighted with a green box. Pathogenic variants (red box), likely pathogenic variants (orange box), and variants of uncertain significance (yellow box) identified in paralogous human tubulins are also shown. D) The effect of identified missense variants on three-dimensional TUBA4A structure. Wild-type residues are coloured green and mutant residues are coloured red. Residues with which the mutant variant shows steric hindrance are purple. Residues which have polar contacts with the wild-type or variant residues are orange. E) A snapshot of mouse TUBA4A-TUBB2A with kinesin (PDB: 8IXF) using Mol*. The GTP molecule (orange) is shown along with TUBA4A (green) and TUBB2A (blue). Gln11 and Ala12 residues are labelled close to the tubulin catalytic GTP site. Gln11 is shown interacting with the GTP molecule. F) Predicted free energy changes (ΔG MUT – ΔG WT ) for missense variants on protein stability. Calculations were performed for variants within very high confidence regions (pLDDT > 90, dark blue bars) or confident regions (70 < pLDDT < 90, light blue bars) of the AlphaFold2-generated TUBA4A protein. Data are given as mean ± s.d. The red dashed lines indicate conservative thresholds (±1.6 kcal/mol). G) Aggrescan 4D prediction plot recreated with Aggrescan 4D scores showing aggregation propensity of wild type TUBA4A and TUBA4A variants including 13 variants identified in the present study for myopathy and multisystem proteinopathy, six variants associated with ALS and two variants previously published in association with ataxia. The x-axis represents the residue position, and the y-axis represents the normalised average aggregation propensity scores calculated by Aggrescan 4D. View this table: View inline View popup Download powerpoint Table 3: Assessment of the thirteen TUBA4A variants observed in our myo-tubulinopathy cohort. AR = Autosomal recessive, AD = Autosomal dominant, n=number of confirmed affected individuals (31), N=number of families (19). One heterozygous variant, p.(Glu284Lys), was identified in three unrelated families and two sporadic cases, with a total of 13 affected individuals harbouring this variant. In two probands, the same substitution, p.(Leu227Phe), was observed, one confirmed to be de novo and the other in a sporadic case. This same substitution was recently identified to have arisen de novo in two myopathy probands in China. 15 Three TUBA4A variants were observed in homozygosity in our myo-tubulinopathy cohort. Firstly, the two families, Pat.21 and the siblings Pat.22 and Pat.23 with the homozygous variants, p.(Ser241Phe) and p.(Gly354Asp), respectively, had disease onset in the first year of life but this was not fatal in the first year. The parents of these three patients were heterozygous carriers and did not manifest any symptoms of neuromuscular disease. In family of Pat. 26, the proband, with an age of onset at 56-60 years, was observed to be homozygous for variant p.(Ala12Thr). His child was found to be heterozygous carrier of the variant and at last clinical assessment had idiopathic epilepsy without any neuromuscular issues. A healthy sibling of the proband had a normal genotype (Supplementary Data 1(available upon request)). All identified variants were in exon 4, except for p.(Gln11His) and p.(Ala12Thr) which were within exon 2. Nine variants were in the guanosine triphosphate (GTP)-ase domain, and the remaining four variants were in the C-terminal domain ( Fig. 3A ). All 13 variants were absent in population databases including gnomADv4.1.0 (Supplementary Data 2). Structural modelling and aggregation analyses reveal variant-specific molecular impacts on TUBA4A Multiple sequence alignments were performed for TUBA4A orthologs across 10 non-human species ( Fig. 3B ), and all human alpha tubulins ( Fig. 3C ), anchored to the human TUBA4A protein sequence. All the identified myo-tubulinopathy variants affected highly conserved amino acid residues. The wild-type TUBA4A protein was predicted by AlphaFold2. The ‘mutagenesis’ function on PyMol was used to predict changes in protein structure and/or folding upon incorporating substituted amino acid residues into the sequence ( Fig. 3D ). Most substitutions showed a change in steric hindrance with nearby or distant amino acid residues. Of note, the two variants observed as de novo , p.(Leu227Phe), p.(Gly350Val) and the homozygous variant p.(Gly354Asp) showed the most substantial changes to polar contacts and steric hindrance upon substitution. Thus, we predicted that these substitutions could affect protein stability. Free energy change analyses showed that, of the identified substitutions, only the p.(Gln11His), p.(Ala12Thr), p.(Leu227Phe), and p.(Gly354Asp) were in the very high confidence regions (pLDDT > 90) of the AlphaFold2-predicted protein structure. FoldX-calculated free energy changes for these substitutions indicated that only the p.(Leu227Phe) substitution has a significant decrease in free energy > 1.6 kcal/mol, suggesting a protein destabilising effect with high-confidence ( Fig. 3F ). This aligns with the previously observed steric hindrance caused by p.(Leu227Phe) ( Fig. 3D ). In addition, the p.(Ser241Phe) substitution showed a significant increase in free energy. This may predict a stability increase, but it cannot be excluded that the variants could cause a deleterious effect to protein folding or stability by another unknown mechanism. Additionally, we used the experimentally validated model of mouse TUBA4A-TUBB2A dimer with kinesin (PDB: 8IXF) to visualise variant impact on the GTP binding site. In the α/β-tubulin heterodimer, both subunits can bind GTP; however, only the β-tubulin subunit is capable of hydrolysing GTP to GDP, while the GTP bound to α-tubulin (TUBA4A) remains non-exchangeable and structurally stabilising. The Gln11 and Ala12 residues in TUBA4A are in proximity to the non-exchangeable GTP-binding site, with Gln11 directly interacting with the nucleotide ( Fig. 3E ). We predict that substitutions at these positions, which alter the residue charge or polarity, may impair GTP binding and thereby destabilise the TUBA4A structure or its interaction with the β-tubulin, thus affecting the microtubule dynamics, an underlying pathomechanisms of different tubulinopathies. Given that structural destabilisation alone may not fully capture the pathogenic potential of the identified variants, we subsequently investigated the aggregation propensity of TUBA4A variants, as stability and aggregation are distinct yet interconnected properties. While FoldX evaluates thermodynamic stability of the native protein fold, aggregation analysis specifically examines the potential for proteins to form multi-protein assemblies with potentially non-native conformations. A variant may significantly destabilise the structure (high ΔΔG in FoldX) without promoting aggregation if it lacks aggregation-prone motifs or is managed by the chaperones. 37 , 38 Conversely, even stable variants can expose or create aggregation-prone regions, altering cellular behaviour. 39 By evaluating both factors, we aimed to clarify how TUBA4A substitutions might contribute to the pathobiology through distinct molecular mechanisms. Aggrescan 4D predicts whether individual residues are aggregation prone (positive score, > 0), have increased solubility (negative score, < 0) or no change in solubility is predicted (0) (Supplementary Data 2). The dynamic mode also predicts the change in interacting residues and the aggregation propensity of the whole protein. Thus, the average aggregation score represents the average of all 448 residues of TUBA4A in both wild-type and each variant protein. All tested variants, including the 13 identified variant residues in our study and the eight previously observed ALS/ataxia-causing variants alter the aggregation propensity of the protein in a variable manner ( Fig. 3G , Supplementary Data 2). These findings suggest that distinct variants, despite differing in predicted stability and aggregation propensity, may modulate TUBA4A behaviour through diverse mechanisms. In-vitro studies show variant-specific disruption of TUBA4A localisation and microtubule morphology To experimentally evaluate the effect of the missense substitutions on intracellular localisation of TUBA4A, COS1 cells were transfected with either WT or TUBA4A variants: p.(Arg105Cys), p.(Thr179Ile), p.(Leu227Phe), p.(Ser241Phe), p.(Glu254Ala), p.(Glu254Gln), p.(Glu254Lys), p.(Glu254Gly), p.(Arg320Cys) and p.(Gly354Asp). Overexpression of wild-type TUBA4A led to an abnormal TUBA4A localisation pattern in ∼53% of the cells. A similar result was seen for p.(Glu254Gly) (∼47%) and p.(Glu254Lys) (∼56%), while p.(Glu254Ala) and p.(Glu254Gln) showed less abnormal cells, ∼23% and ∼26% respectively. A near complete abnormal localisation pattern was observed in ∼87-100% of the cells ( Fig. 4A ) over-expressing p.(Thr179Ile), p.(Leu227Phe), p.(Ser241Phe), p.(Arg320Cys) and p.(Gly354Asp) localisation. Download figure Open in new tab Fig. 4: TUBA4A variants disrupt microtubule morphology in cellular models and patient muscles. A) Molecular physiology of cytoskeleton in COS1 cells overexpressed with TUBA4A-wildtype (wt) and TUBA4A variants. Morphology ranged from normal, mild to abnormal cytoskeleton. As compared to TUBA4A-wt, previously reported disease-causing variants Arg105Cys (ALS) and Arg320Cys (ALS) showed abnormal cytoskeleton structure. Additionally, the myopathic variants identified in our study, Thr179Ile, Leu227Phe, Ser241Phe and Gly354Asp showed abnormal cytoskeletal patterning. Conversely, Glu254Ala, Glu254Gly, Glu254Lys and Gln254Gln showed normal to mildly abnormal cytoskeletal patterning. B) Electron microscopy analysis of microtubules in control muscles and patient muscles showing statistically significant increase in microtubule diameter in patient muscles as compared to controls. Microtubule thickening in myo-tubulinopathies To investigate potential ultrastructural differences in microtubules between patients and age matched controls, we performed additional electron microscopy investigations of patient muscle biopsies from Pat.8 (p.(Glu254Lys), 0-5-year-old male) and Pat.9 (p.(Glu254Gln), 0-5-month-old male) and compared them to two age-matched control biopsies (0-5-year-old males). This analysis revealed significantly thicker microtubules myofibres in patient muscle compared to age-matched healthy controls ( p < 0.0001) ( Fig 4B ). Reassessment of identified TUBA4A variants Based on the genetic data, in silico evidence, and findings from the in vitro assay it was possible to reclassify most of the variants in the myo-tubulinopathy cohort using the ACMG/AMP guidelines ( Table 3 , Supplementary Data 2). 40 Accordingly, classification of eight VUSs could be upgraded to ‘pathogenic’ using additional ACMG/AMP criteria, while there was sufficient evidence to suggest upgrading three variants to ‘likely pathogenic. Two variants had insufficient evidence and remained as VUS ( Table 3 , Supplementary Data 2). Raw and revised classification and assertion criteria details can be seen in Supplementary Data 2. Discussion In this multi-centre study, we identified 13 different missense variants in TUBA4A causing an axial myopathy, with childhood onset of disease being the most frequent clinical presentation. Consistent with our methods of ascertainment, which were largely focused on myopathic cases from neuromuscular disease cohorts, 17/19 families did not have a recorded history of motor neuron disorders, thus this represents an important phenotypic expansion of TUBA4A -related disease to primary skeletal muscle conditions. Two probands presented with multisystem involvement: a mild myofibrillar myopathy along with cerebellar ataxia, cerebellar atrophy and epileptic seizures. These two cases thus represent the first incidence of an MSP phenotype associated with pathogenic variants of TUBA4A . Both MSP associated variants occurred in exon 2 of TUBA4A , whereas the other 11 variants associated with pure myopathic phenotypes were clustered in the exon 4. Three probands harboured homozygous variants in TUBA4A consistent with the observed recessive phenotypes. Of these, two probands presented with severe, early-onset axial myopathy, while the third displayed a later-onset multisystem phenotype. In general, tubulinopathies are either predominantly autosomal dominant (AD) or de novo /sporadic conditions and are rarely autosomal recessive. 41 In our cohort, we observed four families with autosomal dominant inheritance; 12 cases had sporadic occurrence of a skeletal muscle phenotype where five probands had de novo variants in TUBA4A , while in nine isolated cases, we could not clarify the mode of inheritance due to absence of additional familial DNA. Of the remaining families, three showed clear autosomal recessive inheritance with affected individuals homozygous for bi-parentally inherited variants. Notably, multiple substitutions affecting the same amino acid residue resulted in similarly severe myopathic phenotypes in our cohort. Four missense variants were observed at Glu254 (Glu254Gly, Glu254Lys, Glu254Ala and Glu254Gln) in four different probands, each time resulting in a severe early onset phenotype, including two who died in the first year of life. Thus, we observed a range of clinical features including different patterns of inheritance, varying severity of muscle weakness from death in the first year of life (n = 2), non-ambulation in childhood, to mild or moderate weakness in adulthood. In patient muscles biopsies, histopathological findings included characteristic features of a myopathy such as variation in myofibre size, myofibrillar disorganisation, type 1 myofibre predominance, internal nucleation, regenerating and degenerating myofibres, autophagic vacuoles ( n = 4), nemaline bodies ( n = 4), minicores and moth-eaten myofibres. Ultrastructural studies showed classical signs of myofibrillar disarray ( n = 4). Muscle biopsies from the two MSP cases showed similar myopathic features. Owing to the severity of the clinical presentation in affected individuals from the two recessive families, muscle biopsies could not be performed for diagnostic evaluation. In our cohort, however, there were no clear unifying histopathological features. Previously Wan and colleagues 15 showed that in TUBA4A -p.(Leu227Phe) related myopathy, both probands exhibited myofibres with focal myofibrillar disorganisation and rimmed vacuoles. Variable accumulation of phosphorylated TDP-43, often considered a marker of a proteinopathy, showed that different TUBA4A variants might result in distinct level of protein accumulations. Similarly, TUBA4A-positive accumulations were also increased in a subset of muscle biopsies compared to others and controls. Although none of the previous TUBA4A -related ataxia and ALS studies investigated specific autophagic disturbances in patient biopsies, Smith and colleagues 7 showed accumulation of TUBA4A p.(Trp407Ter) in transfected primary motor neurons. Wan and colleagues 15 showed ubiquitin-positive TUBA4A accumulations of the TUBA4A-p.(Leu227Phe) variant in transfected COS7 cells and suggested the involvement of autophagic disturbances. Interestingly, the same variant, p.(Leu227Phe), was observed in two cases within our cohort ( de novo in Pat.7 and as an isolated sporadic occurrence in Pat.6). Complementing these earlier findings, 15 we also observed classical signs of disturbed autophagy, i.e. p62/SQSTM1-positive accumulations in myofibres of affected patients. However, from our immunohistochemistry data and previous studies in tubulin-related pathomechanisms 7 , 42 , protein accumulations may not be the sole contributor to the pathobiology of the disease. We therefore used a range of in silico methods to predict and analyse the molecular impact of identified variants in TUBA4A . Given that tubulin variants can disrupt either tubulin monomer dimerisation or microtubule stability through a dominant-negative mechanism, these computational approaches may offer insights into the distinct ways each variant might contribute to disease pathology. 3D modelling studies indicated that p.(Leu227Phe) showed considerable changes to polar contacts and steric hindrance upon substitution, which could affect protein stability. Further, our FoldX calculations also showed that p.(Leu227Phe) exhibited significantly elevated free energy changes (threshold 1.6 kcal/mol), indicating substantial protein destabilisation. Our in vitro studies in COS1 cells showed abnormal microtubule morphology in cells transfected with the p.(Leu227Phe) variant. These observations are in alignment with Wan and colleagues 15 who showed that the TUBA4A:p.(Leu227Phe) variant forms ubiquitin-positive TUBA4A accumulations in transfected COS7 cells. Notably, while FoldX predicted severe destabilisation, our in-silico aggregation propensity analysis did not suggest an increased tendency toward aggregation for this variant compared to wild-type TUBA4A. This apparent divergence illustrates how distinct molecular mechanisms can lead to similar cellular phenotypes. Our integrated analysis suggests that the p.(Leu227Phe) substitution introduces a bulkier aromatic side chain in a spatially constrained region of TUBA4A, creating specific steric clashes with its neighbouring residues. These structural perturbations directly disrupt the tightly packed hydrophobic interactions that stabilise this domain, accounting for the observed significant increase in free energy. Thus, p.(Leu227Phe) contributes to pathology likely through the thermodynamic destabilisation of the protein’s native fold which may impair proper degradation of the protein resulting in improper localisation and accumulation of the mutant protein rather than through increased aggregation propensity. The p.(Glu254Ala) and the p.(Glu254Gly) variant displayed increased aggregation propensity compared to the wild-type TUBA4A, while our FoldX calculations showed that all identified variants at this hotspot [p.(Glu254Ala), p.(Glu254Lys), p.(Glu254Gln), and p.(Glu254Gly)] have relatively minimal changes in free energy. Interestingly, in vitro over-expression studies revealed normal to mild morphological changes in cells expressing these four variants. This contrasts with the clinical presentation, as patients harbouring variants at this position exhibited the most severe phenotypes in our cohort. Notably, muscle biopsies of Pat.8 with p.(Glu254Lys), Pat.9 with p.(Glu254Gln) and Pat.11 with p.(Glu254Ala) showed TUBA4A positive protein accumulations ( Fig 2B :x-xii). Pat.8 with p.(Glu254Lys), Pat.9 with p.(Glu254Gln) also showed significantly dilated microtubules in skeletal muscle compared to the controls ( Fig 4B ). Again, this divergence between computational predictions, cellular phenotypes, and clinical severity highlights the complex nature of these variants and the limitations of in vitro over-expression assays. While variants at this hotspot may promote aggregation, and possibly impact tubulin dimerisation, the exact pathomechanism underlying the noteworthy clinical impact of this mutational hotspot requires further investigation. Recently, Dodd and colleagues observed similar heterogeneity in tubulin pathologies associated with specific TUBB4B variants, across in silico , in vitro and in vivo studies. 42 The p.(Glu284Lys) variant identified in five unrelated families and 13 affected individuals with similar mild myopathic presentation in our myo-tubulinopathy cohort, presents a notable case of phenotypic divergence. This variant has previously been reported in relation to human zygotic arrest and early developmental failure in heterozygosity. 14 , 43 All cases in our cohort, harbouring this heterozygous variant, either showed autosomal dominant inheritance (Pat.15, Pat.16A, Pat.16B, Pat.17, and Pat.18) or had an undetermined inheritance (Pat.13 and Pat.14), and none of the affected females reported infertility or reproductive complications. Conversely, none of the individuals in the fertility studies 14 , 43 were reported to have neuromuscular disease (personal communication), suggesting potential tissue-specific effects of the variant. However, this pleiotropic effect is not unique to TUBA4A , as similar divergent phenotypes have been observed with other variants such as TUBB3 :p.Arg262His - implicated in cortical malformations, peripheral neuropathy and endocrine malfunction, 44 and LMNA :p.Arg644Cys, which has been associated with muscular dystrophy, cardiomyopathy, insulin resistance, lipodystrophy and peripheral neuropathy. 45 In Pat.21 (p.(Gly354Asp)) and the siblings Pat.22 and Pat.23 (p.(Gly354Asp)) we observed an autosomal recessive myo-tubulinopathy, due to homozygous pathogenic variants. Interestingly, these three patients also presented with fatigability. Repeat nerve stimulation (RNS) showed decrements after repetitive stimulation of ulnar nerve and accessory nerve (Supplementary Data 1(available upon request)). The clinical presentation of fatiguability and decrement on RNS prompted treatment with pyridostigmine. Both patients showed improved muscle function post-treatment. Pat.7 with the de novo p.(Leu227Phe) variant and Pat.25 with the de novo p.(Gly350Val) variant also showed similar fatigability. Pat.7 showed decrement of up to 17% in ulnar nerve and was prescribed salbutamol 0.1 – 0.3 mg/kg/day for 2-3 months but showed no improvement. For Pat.25 there was no decrement observed on RNS of ulnar and ocular muscle but from deltoideus (up to 22%). However, she did not respond to the salbutamol treatment as well. This aligns well with our integrated findings that specific molecular consequences unique to each TUBA4A variant and their distinct downstream interactions may exist in different myo-tubulinopathies, ultimately determining the potential therapeutic approaches and outcomes. In our cohort, 17/19 families did not show a history of a motor neuron disease, and the phenotype observed was primary skeletal muscle disease. However, in these patients, especially with earlier onset and the young ages within the cohort, involvement of CNS cannot be excluded at a later age. Interestingly, in Pat.24 harbouring the heterozygous likely-pathogenic, p.(Gln11His), we observed a mild early-onset cerebellar atrophy along with a myofibrillar myopathy (MFM). In Pat.26 harbouring the homozygous VUS, p.(Ala12Thr), pathologically confirmed MFM and concomitant late-onset ataxia with cerebellar atrophy was observed. The positively charged Gln11 residue interacts with the α-tubulin catalytic GTP site. A change to the negatively charged His at this position will potentially inhibit the GTP binding activity and hence could impact microtubule dynamics. In the α/β-tubulin heterodimer, α-tubulin has a non-hydrolysing and non-exchangeable “N-site” which binds to GTP, whereas β-tubulin has an exchangeable “E-site” where GTP can be hydrolysed into GDP. 46 , 47 Recently, McFadden and colleagues reported the p.(Glu11Arg) variant in TUBB4B , observed in a syndromic presentation of hearing loss, hyperopia without retinal abnormalities, renal tubular Fanconi Syndrome and hypophosphatemic rickets. Functional studies showed that this p.(Glu11Arg) variant results in inhibition of GTPase activity due to the Arg change that impedes depolymerisation and hence results in hyperstability of the polymerised microtubule. 48 The hydrophobic Ala12 residue is close to the α-tubulin GTP binding site, and similarly a change to a polar uncharged Thr residue at this position might impact the GTP binding activity of the heterodimer. Notably, the Gln11His variant was identified as a heterozygous de novo change, whereas the Ala12Thr variant was homozygous. Despite these distinct zygosities, both variants are associated with similar MSP phenotypes, raising intriguing questions about the mechanistic convergence of their allelic presentations. Pat.26 with the homozygous VUS p.(Ala12Thr) was previously published with a VUS in FLNC thought to be the cause of the myofibrillar myopathy in the proband. 49 After the publication, the FLNC variant (p.(Thr2419Met)) has been reported as ‘likely benign’ in multiple submissions in ClinVar (VCV000712192.18). Therefore, with the current knowledge it is unlikely to be the (unique) cause of the disease in the proband, although a potential role in modulating the phenotype cannot be excluded. From our results, we believe that these two substitutions, TUBA4A p.(Gln11His) and p.(Ala12Thr), are quite distinctive in terms of their clinical presentations involving multiple systems, possibly due to the unique biochemical changes to the GTP-binding site in TUBA4A. In general, MSPs encompass a group of inherited disorders characterised by the degeneration of muscle, bone, and nervous tissues. 50 A disease presentation can be classified as an MSP if a neurodegenerative and a neuromuscular phenotype with protein aggregations coexist in the same individual or co-segregate with a variant in the same family. 50 , 51 The shared pathology involves disruptions in key protein clearance pathways, specifically the ubiquitin-proteasome system (UPS) and autophagy 50 - leading to accumulation of toxic protein aggregates, manifesting as diverse clinical phenotypes such as rimmed vacuolar myopathy (RVM), Paget disease of bone (PDB), FTD, and ALS. 50 Essential components of the two major protein quality control pathways: UPS and autophagy, including sequestosome 1/p62 (SQSTM1), 52 play pivotal roles in MSPs. 50 TUBA4A variants have been implicated in neurodegenerative diseases such as ALS, 7 , 53 – 55 FTD, 8 , 9 , 56 and ataxia. 11 In our study, p62 positive accumulations are observed in all patients in whom p62 staining was performed (n = 5). Wan and colleagues also showed presence of p62 positive TUBA4A accumulations in COS-7 cells. 15 The observation of an MSP phenotype in Pat.24 (p.(Gln11His)) and Pat.26 (p.(Ala12Thr)) in our cohort suggests that TUBA4A -related MSPs may represent a unique subtype, warranting further investigation into clinical and molecular features associated with TUBA4A variants observed in MSPs. The diverse neurodegenerative and neuromuscular phenotypes associated with TUBA4A - and tubulins more broadly - can be partly explained by the ‘tubulin code’. This concept proposes that the functional diversity of microtubules, across tissues, is regulated by the expression of distinct tubulin isotypes and a range of post-translational modifications (PTMs), including acetylation, polyglutamylation, and detyrosination. 57 , 58 These PTMs fine-tune microtubule dynamics, stability, and interactions with microtubule-associated proteins, tailoring microtubule behaviour to specific cellular contexts. 59 In skeletal muscle, where microtubules contribute to mechanical stability, nuclear positioning, and metabolic regulation, microtubule regulation is especially critical. 60 Disruption of TUBA4A in this context may therefore have a more pronounced effect on microtubule function, directly impairing myofibre integrity and contractility. Recently, Dodd et al. 42 experimentally showed how the tubulin code explains phenotypic diversity associated with TUBB4B variants. Based on this increasing evidence and our findings, we propose that the tubulin code plays a fundamental role in determining the spectrum of neurodegenerative and neuromuscular manifestations observed in TUBA4A -related disorders, offering a plausible mechanism for the diverse clinical presentations observed. The publicly available GTEx 61 and Human Protein Atlas 62 datasets show ubiquitous expression of alpha-tubulin 4a across human tissues, with notably high expression in skeletal muscle. The wide range of disease onset, variable clinical manifestations and histopathologies, and multiple inheritance patterns observed in the cohort make diagnosis of myo-tubulinopathies and multisystem proteinopathies challenging. Importantly, all disease-causing variants identified in this study were missense changes, many of which would be classified as VUSs under current clinical guidelines owing to limited functional evidence and the absence of an established, reproducible gene-disease relationship for TUBA4A in skeletal muscle disorders. This highlights a critical gap: pathogenic TUBA4A variants may remain unreported or misclassified, delaying diagnosis and hindering appropriate clinical follow-up. In conclusion, this study establishes that missense variants in TUBA4A can cause myopathy and multisystem proteinopathy with variable clinical and histopathological features, including p62 accumulation, internal nucleation, autophagy dysfunction, and myofibrillar disarray. These findings support a role for TUBA4A in maintaining skeletal muscle integrity through the regulation of microtubule function and dynamics. Our large, multi-centre cohort expands the phenotypic and inheritance spectrum of TUBA4A -related disease, with most patients showing no evidence of CNS involvement. Together, these results underscore the importance of including TUBA4A in genetic screening panels for unresolved myopathies and related neuromuscular conditions. Data Availability All relevant imaging and biological materials included in this study are available from the authors (or commercial providers). Sensitive sr-ES/sr-GS data of patients and family members are deposited in seqr and RD-Connect GPAP. Funding This work is supported by The Fred Liuzzi Foundation (G.R./M.J.), the Association Française contre les Myopathies (AFM Téléthon, The French Muscular Dystrophy Association, grant award number: 24438, M.J.), the Australian Medical Research Futures Fund (MRFF)-Genomics Health Futures Mission Grant (APP2007681, N.G.L), the Australian MRFF Grant (APP2023357, G.R.). M.T. is supported by Italian Ministry of Health (Current Research Funds, RF-2021-12374963). G.R. is supported by an Australian NHMRC Fellowship (APP2007769). J.M.P. and C.F. are supported by the Australian Government research training program scholarship. C.F. is additionally, supported by Jean Rogerson HDR Scholarship and the Jock and Marjorie Hetherington HDR Top-Up Scholarship. Sequencing and analysis of Pat. 8 (BOS1139-1) was supported by the Broad Institute of MIT and Harvard Center for Mendelian Genomics (Broad CMG) funded by U01HG011755 from the NHGRI, the Boston Children’s Hospital IDDRC Molecular Genetics Core Facility funded by P50HD105351 from the NICHD, and as part of the Boston Children’s Hospital Children’s Rare Disease Collaborative study. Sequencing and analysis of patients 14 and 15 were provided by the Broad Institute of MIT and Harvard Center for Mendelian Genomics (Broad CMG) and were funded by the National Human Genome Research Institute grants, UM1 HG008900 (with additional support from the National Eye Institute, and the National Heart, Lung and Blood Institute), U01 HG011755 and R01 HG009141, and by the Chan Zuckerberg Initiative Donor-Advised Fund at the Silicon Valley Community Foundation, 2022-309464 and 2019-199278 ( https://doi.org/10.37921/236582yuakxy ) (funder DOI 10.13039/100014989). Patients 16 and 19 were sequenced as part of the MYO-SEQ project, which was funded by Sanofi Genzyme, Ultragenyx, LGMD2I Research Fund, Samantha J. Brazzo Foundation, LGMD2D Foundation and Kurt+Peter Foundation, Muscular Dystrophy UK, and Coalition to Cure Calpain 3. M.S. received fundings from the European Union (Grant Agreement 101080844 to CoMPaSS-NMD), Academy of Finland (grant 339434, 346209, 361979), Association Française contre les Myopathies (grant 23281), Sigrid Juselius Foundation (230217), and Samfundet Folkhälsan i Svenska Finland. GT is supported by the Academy of Medical Sciences Professorship Scheme (Grant APR8\1017). Competing interests The authors report no competing interests. Author contributions Conceptualisation of the study: MJ, GR Project administration: MJ, GR Funding acquisition: MJ, GR, NGL Patient samples and clinical/genomic data collection: All clinicians and molecular geneticists Data analysis and curation: MJ, CF, JMP, MMO, YS, MA, EYZ, HRL, CCB, SAS, EAE, LMK, PBK, IOO, BM, LP, MOL, CAT, AODL, FCL, ADA, AC, MT, AP, CVG, GS, MLMN, MC, CAG, ZMV, AHB, VM, GK, AK, AT, NSP, JV, CGC, EZ, CMM, MS, GT, VS, BU, IN, GR Methodology: MJ, YS, MMO, JMP, GR Visualisation: MJ, YS, MMO, JMP, KSS, IAC, DA, SK, CMM, GS, NSP, JV Writing the original draft: MJ, YS, MMO, JMP, GR Review and editing of the manuscript: All authors View this table: View inline View popup Download powerpoint Supplementary Table Assessment of variants in TUBA4A observed in our cohort. AL=Acceptor Loss, AG=Acceptor Gain, AR = Autosomal recessive, AD = Autosomal dominant, n=number of confirmed affected individuals, N=number of families, NA=Not assessed. Acknowledgements The authors would like to thank the patients and their families for cooperation in this study. Wathone Win and Ronald van Beek for technical assistance. Clinical and genetic data of the most patients were shared in RD-Connect, which received funding from the European Union Seventh Framework Programme (FP7/2007-2013) under grant agreement No. 305444 within the Solve-RD project. Solve-RD project has received funding from the European Union’s Horizon 2020 research and innovation programme under grant agreement No 779257. This work was supported by resources provided by the Pawsey Supercomputing Research Centre with funding from the Australian Government and the Government of Western Australia. References 1. ↵ Bahi-Buisson N , Maillard C. Tubulinopathies Overview . In: Adam MP , Feldman J , Mirzaa GM , Pagon RA , Wallace SE , Amemiya A , eds. GeneReviews((R)) . University of Washington , Seattle Copyright © 1993-2024, University of Washington, Seattle. GeneReviews is a registered trademark of the University of Washington, Seattle. All rights reserved.; 1993 . 2. ↵ Desai A , Mitchison TJ . Microtubule polymerization dynamics . Annu Rev Cell Dev Biol . 1997 ; 13 : 83 – 117 . doi: 10.1146/annurev.cellbio.13.1.83 OpenUrl CrossRef PubMed Web of Science 3. Mitchison T , Kirschner M . Dynamic instability of microtubule growth . Nature . Nov 15–21 1984 ; 312 ( 5991 ): 237 – 42 . doi: 10.1038/312237a0 OpenUrl CrossRef PubMed Web of Science 4. ↵ Guzik BW , Goldstein LS . Microtubule-dependent transport in neurons: steps towards an understanding of regulation, function and dysfunction . Curr Opin Cell Biol . Aug 2004 ; 16 ( 4 ): 443 – 50 . doi: 10.1016/j.ceb.2004.06.002 OpenUrl CrossRef PubMed Web of Science 5. ↵ Romaniello R , Arrigoni F , Fry AE , et al. Tubulin genes and malformations of cortical development . Eur J Med Genet . Dec 2018 ; 61 ( 12 ): 744 – 754 . doi: 10.1016/j.ejmg.2018.07.012 OpenUrl CrossRef PubMed 6. ↵ Montecinos-Franjola F , Chaturvedi SK , Schuck P , Sackett DL . All tubulins are not alike: Heterodimer dissociation differs among different biological sources . J Biol Chem . Jun 28 2019 ; 294 ( 26 ): 10315 – 10324 . doi: 10.1074/jbc.RA119.007973 OpenUrl Abstract / FREE Full Text 7. ↵ Smith BN , Ticozzi N , Fallini C , et al. Exome-wide rare variant analysis identifies TUBA4A mutations associated with familial ALS . Neuron . Oct 22 2014 ; 84 ( 2 ): 324 – 31 . doi: 10.1016/j.neuron.2014.09.027 OpenUrl CrossRef PubMed Web of Science 8. ↵ Mol MO , Wong TH , Melhem S , et al. Novel TUBA4A Variant Associated With Familial Frontotemporal Dementia . Neurol Genet . Jun 2021 ; 7 ( 3 ): e596 . doi: 10.1212/NXG.0000000000000596 OpenUrl Abstract / FREE Full Text 9. ↵ Van Schoor E , Vandenbulcke M , Bercier V , et al. Frontotemporal Lobar Degeneration Case with an N-Terminal TUBA4A Mutation Exhibits Reduced TUBA4A Levels in the Brain and TDP-43 Pathology . Biomolecules . Mar 12 2022 ; 12 (3) doi: 10.3390/biom12030440 OpenUrl CrossRef 10. ↵ Okada K , Hata Y , Ichimata S , et al. An autopsy case of pure nigropathy with TUBA4A nonsense mutation . Neuropathol Appl Neurobiol . Oct 2021 ; 47 ( 6 ): 891 – 893 . doi: 10.1111/nan.12712 OpenUrl CrossRef PubMed 11. ↵ Torella A , Ricca I , Piluso G , et al. A new genetic cause of spastic ataxia: the p.Glu415Lys variant in TUBA4A . J Neurol . Oct 2023 ; 270 ( 10 ): 5057 – 5063 . doi: 10.1007/s00415-023-11816-w OpenUrl CrossRef 12. ↵ Benkirane M , Bonhomme M , Morsy H , et al. De novo and inherited monoallelic variants in TUBA4A cause ataxia and spasticity . Brain . Nov 4 2024 ; 147 ( 11 ): 3681 – 3689 . doi: 10.1093/brain/awae193 OpenUrl CrossRef PubMed 13. ↵ Hu H , Wan X , Zhang H , et al. Biallelic variants in α-tubulin isotypes cause female infertility characterised as recurrent preimplantation embryo arrest . J Med Genet . Oct 23 2024 ; 61 ( 11 ): 1045 – 1052 . doi: 10.1136/jmg-2024-110163 OpenUrl Abstract / FREE Full Text 14. ↵ Li L , Mao L , Zhang Z , et al. Mutations in TUBA4A lead to human zygotic arrest and early developmental failure . Life Medicine . 2023 ; 2 (5) doi: 10.1093/lifemedi/lnad032 OpenUrl CrossRef 15. ↵ Wan Y , Zhou C , Chang X , et al. Novel TUBA4A variant causes congenital myopathy with focal myofibrillar disorganisation . J Med Genet . Jun 20 2024 ; 61 ( 7 ): 626 – 632 . doi: 10.1136/jmg-2023-109786 OpenUrl Abstract / FREE Full Text 16. ↵ Hoff KJ , Aiken JE , Gutierrez MA , Franco SJ , Moore JK . TUBA1A tubulinopathy mutants disrupt neuron morphogenesis and override XMAP215/Stu2 regulation of microtubule dynamics . Elife . May 5 2022 ; 11 doi: 10.7554/eLife.76189 OpenUrl CrossRef 17. Hoff KJ , Neumann AJ , Moore JK . The molecular biology of tubulinopathies: Understanding the impact of variants on tubulin structure and microtubule regulation . Front Cell Neurosci . 2022 ; 16 : 1023267 . doi: 10.3389/fncel.2022.1023267 OpenUrl CrossRef PubMed 18. Jurgens JA , Barry BJ , Lemire G , et al. Novel variants in TUBA1A cause congenital fibrosis of the extraocular muscles with or without malformations of cortical brain development . Eur J Hum Genet . May 2021 ; 29 ( 5 ): 816 – 826 . doi: 10.1038/s41431-020-00804-7 OpenUrl CrossRef PubMed 19. ↵ Puri D , Barry BJ , Engle EC . TUBB3 and KIF21A in neurodevelopment and disease . Front Neurosci . 2023 ; 17 : 1226181 . doi: 10.3389/fnins.2023.1226181 OpenUrl CrossRef PubMed 20. ↵ Perrone F , Nguyen HP , Van Mossevelde S , et al. Investigating the role of ALS genes CHCHD10 and TUBA4A in Belgian FTD-ALS spectrum patients . Neurobiol Aging . Mar 2017 ; 51 : 177 e9 – 177 e16. doi: 10.1016/j.neurobiolaging.2016.12.008 OpenUrl CrossRef 21. ↵ Dubowitz V , Oldfors A , Sewry CA. Muscle biopsy : A Practical Approach . Saunders ,. 22. ↵ Pais LS , Snow H , Weisburd B , et al. seqr: A web-based analysis and collaboration tool for rare disease genomics . Hum Mutat . Jun 2022 ; 43 ( 6 ): 698 – 707 . doi: 10.1002/humu.24366 OpenUrl CrossRef PubMed 23. ↵ Laurie S , Piscia D , Matalonga L , et al. The RD-Connect Genome-Phenome Analysis Platform: Accelerating diagnosis, research, and gene discovery for rare diseases . Hum Mutat . Jun 2022 ; 43 ( 6 ): 717 – 733 . doi: 10.1002/humu.24353 OpenUrl CrossRef PubMed 24. ↵ Karczewski KJ , Francioli LC , Tiao G , et al. The mutational constraint spectrum quantified from variation in 141,456 humans . Nature . May 2020 ; 581 ( 7809 ):434-443. doi: 10.1038/s41586-020-2308-7 OpenUrl CrossRef PubMed 25. ↵ Chen S , Francioli LC , Goodrich JK , et al. A genomic mutational constraint map using variation in 76,156 human genomes . Nature . Jan 2024 ; 625 ( 7993 ):92-100. doi: 10.1038/s41586-023-06045-0 OpenUrl CrossRef PubMed 26. ↵ Kopanos C , Tsiolkas V , Kouris A , et al. VarSome: the human genomic variant search engine . Bioinformatics . Jun 1 2019 ; 35 ( 11 ): 1978 – 1980 . doi: 10.1093/bioinformatics/bty897 OpenUrl CrossRef PubMed 27. ↵ Delgado J , Radusky LG , Cianferoni D , Serrano L . FoldX 5.0: working with RNA, small molecules and a new graphical interface . Bioinformatics . Oct 15 2019 ; 35 ( 20 ): 4168 – 4169 . doi: 10.1093/bioinformatics/btz184 OpenUrl CrossRef PubMed 28. ↵ Akdel M , Pires DEV , Pardo EP , et al. A structural biology community assessment of AlphaFold2 applications . Nat Struct Mol Biol . Nov 2022 ; 29 ( 11 ): 1056 – 1067 . doi: 10.1038/s41594-022-00849-w OpenUrl CrossRef PubMed 29. ↵ Ravenscroft G , Miyatake S , Lehtokari VL , et al. Mutations in KLHL40 are a frequent cause of severe autosomal-recessive nemaline myopathy . Am J Hum Genet . Jul 11 2013 ; 93 ( 1 ): 6 – 18 . doi: 10.1016/j.ajhg.2013.05.004 OpenUrl CrossRef PubMed 30. ↵ Mao K , Borel C , Ansar M , et al. FOXI3 pathogenic variants cause one form of craniofacial microsomia . Nat Commun . Apr 11 2023 ; 14 ( 1 ): 2026 . doi: 10.1038/s41467-023-37703-6 OpenUrl CrossRef PubMed 31. ↵ Badaczewska-Dawid AE , Kuriata A , Pintado-Grima C , et al. A3D Model Organism Database (A3D-MODB): a database for proteome aggregation predictions in model organisms . Nucleic Acids Res . Jan 5 2024 ; 52 ( D1 ): D360 – D367 . doi: 10.1093/nar/gkad942 OpenUrl CrossRef PubMed 32. ↵ Kuriata A , Iglesias V , Pujols J , Kurcinski M , Kmiecik S , Ventura S . Aggrescan3D (A3D) 2.0: prediction and engineering of protein solubility . Nucleic Acids Res . Jul 2 2019 ; 47 ( W1 ): W300 – W307 . doi: 10.1093/nar/gkz321 OpenUrl CrossRef 33. ↵ Zalewski M , Iglesias V , Barcenas O , Ventura S , Kmiecik S . Aggrescan4D: A comprehensive tool for pH-dependent analysis and engineering of protein aggregation propensity . Protein Sci . Oct 2024 ; 33 ( 10 ): e5180 . doi: 10.1002/pro.5180 OpenUrl CrossRef PubMed 34. ↵ Jumper J , Evans R , Pritzel A , et al. Highly accurate protein structure prediction with AlphaFold . Nature . Aug 2021 ; 596 ( 7873 ):583-589. doi: 10.1038/s41586-021-03819-2 OpenUrl CrossRef PubMed 35. ↵ Diao L , Zheng W , Zhao Q , et al. Cryo-EM of α-tubulin isotype-containing microtubules revealed a contracted structure of α4A/β2A microtubules . Acta Biochim Biophys Sin (Shanghai) . Oct 25 2023 ; 55 ( 10 ): 1551 – 1560 . doi: 10.3724/abbs.2023130 OpenUrl CrossRef PubMed 36. ↵ Sehnal D , Bittrich S , Deshpande M , et al. Mol* Viewer: modern web app for 3D visualization and analysis of large biomolecular structures . Nucleic Acids Res . Jul 2 2021 ; 49 ( W1 ): W431 – w437 . doi: 10.1093/nar/gkab314 OpenUrl CrossRef PubMed 37. ↵ de Rosa M , Barbiroli A , Bonì F , et al. The structure of N184K amyloidogenic variant of gelsolin highlights the role of the H-bond network for protein stability and aggregation properties . Eur Biophys J . Jan 2020 ; 49 ( 1 ): 11 – 19 . doi: 10.1007/s00249-019-01409-9 OpenUrl CrossRef PubMed 38. ↵ Das P , Murray B , Belfort G . Alzheimer’s protective A2T mutation changes the conformational landscape of the Aβ₁₋₄₂ monomer differently than does the A2V mutation . Biophys J . Feb 3 2015 ; 108 ( 3 ): 738 – 47 . doi: 10.1016/j.bpj.2014.12.013 OpenUrl CrossRef PubMed 39. ↵ Tartaglia GG , Pawar AP , Campioni S , Dobson CM , Chiti F , Vendruscolo M . Prediction of aggregation-prone regions in structured proteins . J Mol Biol . Jul 4 2008 ; 380 ( 2 ): 425 – 36 . doi: 10.1016/j.jmb.2008.05.013 OpenUrl CrossRef PubMed Web of Science 40. ↵ Richards S , Aziz N , Bale S , et al. Standards and guidelines for the interpretation of sequence variants: a joint consensus recommendation of the American College of Medical Genetics and Genomics and the Association for Molecular Pathology . Genet Med . May 2015 ; 17 ( 5 ): 405 – 24 . doi: 10.1038/gim.2015.30 OpenUrl CrossRef PubMed 41. ↵ Abdollahi MR , Morrison E , Sirey T , et al. Mutation of the variant alpha-tubulin TUBA8 results in polymicrogyria with optic nerve hypoplasia . Am J Hum Genet . Nov 2009 ; 85 ( 5 ): 737 – 44 . doi: 10.1016/j.ajhg.2009.10.007 OpenUrl CrossRef PubMed Web of Science 42. ↵ Dodd DO , Mechaussier S , Yeyati PL , et al. Ciliopathy patient variants reveal organelle-specific functions for TUBB4B in axonemal microtubules . Science . Apr 26 2024 ; 384 ( 6694 ): eadf5489 . doi: 10.1126/science.adf5489 OpenUrl CrossRef PubMed 43. ↵ Li Q , Zhao L , Zeng Y , et al. Large-scale analysis of de novo mutations identifies risk genes for female infertility characterized by oocyte and early embryo defects . Genome Biol . Apr 6 2023 ; 24 ( 1 ): 68 . doi: 10.1186/s13059-023-02894-0 OpenUrl CrossRef PubMed 44. ↵ Whitman MC , Barry BJ , Robson CD , et al. TUBB3 Arg262His causes a recognizable syndrome including CFEOM3, facial palsy, joint contractures, and early-onset peripheral neuropathy . Human Genetics . 2021/12/01 2021 ; 140 ( 12 ): 1709 - 1731 . doi: 10.1007/s00439-021-02379-9 OpenUrl CrossRef PubMed 45. ↵ Storey EC , Fuller HR . Genotype-Phenotype Correlations in Human Diseases Caused by Mutations of LINC Complex-Associated Genes: A Systematic Review and Meta-Summary . Cells . 2022 ; 11 ( 24 ). doi: 10.3390/cells11244065 OpenUrl CrossRef PubMed 46. ↵ Jacobs M , Smith H , Taylor EW . Tublin: nucleotide binding and enzymic activity . J Mol Biol . Nov 5 1974 ; 89 ( 3 ): 455 – 68 . doi: 10.1016/0022-2836(74)90475-6 OpenUrl CrossRef PubMed 47. ↵ Brylawski BP , Caplow M . Rate for nucleotide release from tubulin . J Biol Chem . Jan 25 1983 ; 258 ( 2 ): 760 – 3 . OpenUrl Abstract / FREE Full Text 48. ↵ McFadden JR , Tolete CDP , Huang Y , et al. Clinical, genetic, and structural characterization of a novel TUBB4B tubulinopathy . Mol Genet Metab Rep . Sep 2023 ; 36 : 100990 . doi: 10.1016/j.ymgmr.2023.100990 OpenUrl CrossRef 49. ↵ Tasca G , Odgerel Z , Monforte M , et al. Novel FLNC mutation in a patient with myofibrillar myopathy in combination with late-onset cerebellar ataxia . Muscle & Nerve . 2012 ; 46 ( 2 ): 275 – 282 . doi: 10.1002/mus.23349 OpenUrl CrossRef PubMed 50. ↵ Korb MK , Kimonis VE , Mozaffar T . Multisystem proteinopathy: Where myopathy and motor neuron disease converge . Muscle Nerve . Apr 2021 ; 63 ( 4 ): 442 – 454 . doi: 10.1002/mus.27097 OpenUrl CrossRef PubMed 51. ↵ Taylor JP . Multisystem proteinopathy: intersecting genetics in muscle, bone, and brain degeneration . Neurology . Aug 25 2015 ; 85 ( 8 ): 658 – 60 . doi: 10.1212/WNL.0000000000001862 OpenUrl CrossRef PubMed 52. ↵ Bjorkoy G , Lamark T , Brech A , et al. p62/SQSTM1 forms protein aggregates degraded by autophagy and has a protective effect on huntingtin-induced cell death . J Cell Biol . Nov 21 2005 ; 171 ( 4 ): 603 – 14 . doi: 10.1083/jcb.200507002 OpenUrl Abstract / FREE Full Text 53. ↵ Rademakers R , van Blitterswijk M . Excess of rare damaging TUBA4A variants suggests cytoskeletal defects in ALS . Neuron . Oct 22 2014 ; 84 ( 2 ): 241 – 3 . doi: 10.1016/j.neuron.2014.10.002 OpenUrl CrossRef PubMed 54. Li J , He J , Tang L , Chen L , Ma Y , Fan D . Screening for TUBA4A mutations in a large Chinese cohort of patients with ALS: re-evaluating the pathogenesis of TUBA4A in ALS . J Neurol Neurosurg Psychiatry . Dec 2018 ; 89 ( 12 ): 1350 – 1352 . doi: 10.1136/jnnp-2017-317560 OpenUrl FREE Full Text 55. ↵ Ganne A , Balasubramaniam M , Ayyadevara H , et al. In silico analysis of TUBA4A mutations in Amyotrophic Lateral Sclerosis to define mechanisms of microtubule disintegration . Sci Rep . Feb 6 2023 ; 13 ( 1 ): 2096 . doi: 10.1038/s41598-023-28381-x OpenUrl CrossRef PubMed 56. ↵ Dols-Icardo O , Iborra O , Valdivia J , et al. Assessing the role of TUBA4A gene in frontotemporal degeneration . Neurobiol Aging . Feb 2016 ; 38 : 215 e13 – 215 e14. doi: 10.1016/j.neurobiolaging.2015.10.030 OpenUrl CrossRef 57. ↵ Janke C , Magiera MM . The tubulin code and its role in controlling microtubule properties and functions . Nat Rev Mol Cell Biol . Jun 2020 ; 21 ( 6 ): 307 – 326 . doi: 10.1038/s41580-020-0214-3 OpenUrl CrossRef PubMed 58. ↵ Verhey KJ , Gaertig J. The tubulin code . Cell Cycle . Sep 1 2007 ; 6 ( 17 ): 2152 - 60 . doi: 10.4161/cc.6.17.4633 OpenUrl CrossRef PubMed Web of Science 59. ↵ 59. Ludueña RF. Multiple Forms of Tubulin: Different Gene Products and Covalent Modifications . In: Jeon KW , ed. International Review of Cytology . Academic Press ; 1997 : 207 – 275 . 60. ↵ Denes LT , Kelley CP , Wang ET . Microtubule-based transport is essential to distribute RNA and nascent protein in skeletal muscle . Nat Commun . Oct 27 2021 ; 12 ( 1 ): 6079 . doi: 10.1038/s41467-021-26383-9 OpenUrl CrossRef PubMed 61. ↵ Lonsdale J , Thomas J , Salvatore M , et al. The Genotype-Tissue Expression (GTEx) project . Nature Genetics . 2013/06/01 2013 ; 45 ( 6 ): 580 – 585 . doi: 10.1038/ng.2653 OpenUrl CrossRef PubMed 62. ↵ Uhlén M , Fagerberg L , Hallström BM , et al. Tissue-based map of the human proteome . Science . 2015 ; 347 ( 6220 ): 1260419 . doi : doi: 10.1126/science.1260419 OpenUrl Abstract / FREE Full Text View the discussion thread. Back to top Previous Next Posted June 28, 2025. Download PDF Data/Code Email Thank you for your interest in spreading the word about medRxiv. NOTE: Your email address is requested solely to identify you as the sender of this article. Your Email * Your Name * Send To * Enter multiple addresses on separate lines or separate them with commas. You are going to email the following Missense variants in TUBA4A cause myo-tubulinopathies Message Subject (Your Name) has forwarded a page to you from medRxiv Message Body (Your Name) thought you would like to see this page from the medRxiv website. Your Personal Message CAPTCHA This question is for testing whether or not you are a human visitor and to prevent automated spam submissions. Share Missense variants in TUBA4A cause myo-tubulinopathies Mridul Johari , Chiara Folland , Yoshihiko Saito , Machteld M. Oud , Jevin M. Parmar , Ana Töpf , Sergei Kurbatov , Maria Ampleeva , Ekaterina Y. Zakharova , Irina A. Chekmareva , Ksenia S. Shirokova , Dmitrii Atiakshin , Thatjana Gardeitchik , Erik-Jan Kamsteeg , Evita Medici , Laura Donker Kaat , Christine C. Bruels , Seth A. Stafki , Elicia A. Estrella , Hannah R. Littel , Louis M. Kunkel , Peter B. Kang , Ikeoluwa Osei-Owusu , Lynn Pais , Melaine O’Leary , Christina Austin-Tse , Anne O’Donnell-Luria , Brian Mangilog , Francesca Clementina Radio , Adele D’Amico , Andrea Ciolfi , Marco Tartaglia , Aurélien Perrin , Charles Van Goethem , Guilhem Sole , Marie-Laure Martin-Négrier , Mireille Cossée , Casie A. Genetti , Zaheer M. Valivullah , Vedrana Milic , Gordana Kovacevic , Ana Kosac , Cristiane A.M. Moreno , Clara Gontijo Camelo , Edmar Zanoteli , Michael C. Fahey , Alan H. Beggs , John Vissing , Volker Straub , Marco Savarese , Giorgio Tasca , Nicol Voermans , Nigel G. Laing , Bjarne Udd , Ichizo Nishino , Gianina Ravenscroft medRxiv 2025.06.26.25330266; doi: https://doi.org/10.1101/2025.06.26.25330266 Share This Article: Copy Citation Tools Missense variants in TUBA4A cause myo-tubulinopathies Mridul Johari , Chiara Folland , Yoshihiko Saito , Machteld M. Oud , Jevin M. Parmar , Ana Töpf , Sergei Kurbatov , Maria Ampleeva , Ekaterina Y. Zakharova , Irina A. Chekmareva , Ksenia S. Shirokova , Dmitrii Atiakshin , Thatjana Gardeitchik , Erik-Jan Kamsteeg , Evita Medici , Laura Donker Kaat , Christine C. Bruels , Seth A. Stafki , Elicia A. Estrella , Hannah R. Littel , Louis M. Kunkel , Peter B. Kang , Ikeoluwa Osei-Owusu , Lynn Pais , Melaine O’Leary , Christina Austin-Tse , Anne O’Donnell-Luria , Brian Mangilog , Francesca Clementina Radio , Adele D’Amico , Andrea Ciolfi , Marco Tartaglia , Aurélien Perrin , Charles Van Goethem , Guilhem Sole , Marie-Laure Martin-Négrier , Mireille Cossée , Casie A. Genetti , Zaheer M. Valivullah , Vedrana Milic , Gordana Kovacevic , Ana Kosac , Cristiane A.M. Moreno , Clara Gontijo Camelo , Edmar Zanoteli , Michael C. Fahey , Alan H. Beggs , John Vissing , Volker Straub , Marco Savarese , Giorgio Tasca , Nicol Voermans , Nigel G. Laing , Bjarne Udd , Ichizo Nishino , Gianina Ravenscroft medRxiv 2025.06.26.25330266; doi: https://doi.org/10.1101/2025.06.26.25330266 Citation Manager Formats BibTeX Bookends EasyBib EndNote (tagged) EndNote 8 (xml) Medlars Mendeley Papers RefWorks Tagged Ref Manager RIS Zotero Tweet Widget Facebook Like Google Plus One Subject Area Neurology Subject Areas All Articles Addiction Medicine (568) Allergy and Immunology (863) Anesthesia (297) Cardiovascular Medicine (4420) Dentistry and Oral Medicine (443) Dermatology (381) Emergency Medicine (606) Endocrinology (including Diabetes Mellitus and Metabolic Disease) (1507) Epidemiology (15212) Forensic Medicine (30) Gastroenterology (1120) Genetic and Genomic Medicine (6581) Geriatric Medicine (667) Health Economics (996) Health Informatics (4519) Health Policy (1366) Health Systems and Quality Improvement (1611) Hematology (538) HIV/AIDS (1264) Infectious Diseases (except HIV/AIDS) (15906) Intensive Care and Critical Care Medicine (1103) Medical Education (620) Medical Ethics (144) Nephrology (667) Neurology (6580) Nursing (345) Nutrition (998) Obstetrics and Gynecology (1141) Occupational and Environmental Health (956) Oncology (3323) Ophthalmology (970) Orthopedics (369) Otolaryngology (420) Pain Medicine (435) Palliative Medicine (129) Pathology (663) Pediatrics (1689) Pharmacology and Therapeutics (691) Primary Care Research (710) Psychiatry and Clinical Psychology (5429) Public and Global Health (9212) Radiology and Imaging (2192) Rehabilitation Medicine and Physical Therapy (1368) Respiratory Medicine (1194) Rheumatology (593) Sexual and Reproductive Health (709) Sports Medicine (529) Surgery (709) Toxicology (99) Transplantation (288) Urology (265) (function(){function c(){var b=a.contentDocument||a.contentWindow.document;if(b){var d=b.createElement('script');d.innerHTML="window.__CF$cv$params={r:'9fefbd785add300f',t:'MTc3OTMyNjIzMg=='};var a=document.createElement('script');a.src='/cdn-cgi/challenge-platform/scripts/jsd/main.js';document.getElementsByTagName('head')[0].appendChild(a);";b.getElementsByTagName('head')[0].appendChild(d)}}if(document.body){var a=document.createElement('iframe');a.height=1;a.width=1;a.style.position='absolute';a.style.top=0;a.style.left=0;a.style.border='none';a.style.visibility='hidden';document.body.appendChild(a);if('loading'!==document.readyState)c();else if(window.addEventListener)document.addEventListener('DOMContentLoaded',c);else{var e=document.onreadystatechange||function(){};document.onreadystatechange=function(b){e(b);'loading'!==document.readyState&&(document.onreadystatechange=e,c())}}}})();