TUBB4B variants specifically impact ciliary function, causing a ciliopathic spectrum

Cilia are small microtubule-based structures found on the surface of most mammalian cells, which have key sensory and sometimes motile functions. Primary ciliary dyskinesia (PCD) is a type of ciliopathy caused by defects in motile cilia. The genetic basis of PCD is only partially understood. Studying a cohort of 11 human patients with PCD, we find that de novo mutations in TUBB4B , a beta tubulin isotype, cause three distinct classes of ciliopathic disease. In vivo studies in mice show that Tubb4b plays a specific role in cilia, building centrioles and axonemes in multiciliated cells. Examining the effects of specific TUBB4B variants in cells and in mice, we further demonstrate that distinct TUBB4B mutations differentially affect microtubule dynamics and cilia formation in a dominant negative manner. Finally, structure-function studies reveal that different TUBB4B mutations disrupt distinct tubulin interfaces. Importantly, these molecular differences correlate with disease features. We show that tubulin heterodimer-impairing TUBB4B variants underlie nonsyndromic PCD, whilst additional renal and sensorineural ciliopathic features in a syndromic PCD subtype arise from microtubule lumenal interface-impaired TUBB4B variants. These findings suggest that specific tubulin isotypes have distinct and non-redundant subcellular functions, and demonstrate that human tubulinopathies can be drivers of ciliopathic syndromes. ### Competing Interest Statement The authors have declared no competing interest. ### Funding Statement We are grateful for support from the MRC (PY, FM, PT, LM, PM: MC\_UU\_12018/26). This project has received funding from the European Research Council (ERC) under the European Union's Horizon 2020 research and innovation programme (grant agreement no.866355: DD, PT, PM). Funding support for this project was also provided as an MRC Career Development Award (MR/M02122X/1) and Lister Prize Fellowship to JAM; an NHS Research Scotland fellowship to SU; and an NRS/R+D fellowship from the NHS Lothian R&D office to DU. SA is supported by MRC Core funding to the MRC Human Genetics Unit (MRC grant MC\_UU\_00007/16). The Scottish Genomes Partnership is funded by the Chief Scientist Office of the Scottish Government Health Directorates [SGP/1] and the Medical Research Council Whole Genome Sequencing for Health and Wealth Initiative (MC/PC/15080). The MS work was funded by the Wellcome Trust (Multiuser Equipment Grant, 208402/Z/17/Z). This work was supported by grants from the Fondation de France Berthe Fouassier (Engt 00079330) and Association Autour de Faustine to SM, Retina France; Fondation JED Belgique; Union Nationale des Aveugles et Deficients Visuels (UNADEV)-Alliance Nationale pour les sciences de la vie et de la sante (AVIESAN) (R16073KS), and Fondation Visio to IP and JMR. JMR is member of the European Reference Network for Rare Eye Diseases (ERN-EYE), which is co-funded by the Health Program of the European Union under the Framework Partnership Agreement no.739534. Funding support for research was provided to MRK, MWL, MAZ and MR by US NIH/ORDR/NACTS/NHLBI grant U54HL096458; and to MRK, MAZ by US NIH/NHLBI grant R01HL071798. The Genetic Disorders of Mucociliary Clearance Consortium (U54HL096458) is part of the National Center for Advancing Translational Sciences (NCATS) Rare Diseases Clinical Research Network (RDCRN) and supported by the RDCRN Data Management and Coordinating Center (DMCC) (U2CTR002818). RDCRN is an initiative of the Office of Rare Diseases Research (ORDR) funded through a collaboration between NCATS and National Heart, Lung, and Blood Institute (NHLBI). The Yale Center for Mendelian Genomics (UM1HG006504) is funded by the US NIH/NHGRI (National Human Genome Research Institute). The contents are solely the responsibility of the authors and do not necessarily represent the official views of the National Institute of Health. HMM is supported by NIHR GOSH BRC, Ministry of Higher Education in Egypt, MRF is supported by a Wellcome Trust Collaborative Award in Science (210585/Z/18/Z). HMM, SC, AS, CH acknowledge support from the BEAT-PCD network (COST Action 1407 and European Respiratory Society (ERS) Clinical Research Collaboration). Research reported in this manuscript was supported by the NIH Common Fund, through the Office of Strategic Coordination/Office of the NIH Director under Award Number U01HG007690. The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Institutes of Health. MG is supported by a Charles A. King Trust Postdoctoral Research Fellowship. AB is supported by NIGMS grant GM141109 and the Pew Charitable Trusts. JW is supported by a Wellcome Senior Research Fellowship (207430). TA is supported by an MRC studentship (MR/N013166/1). The Wellcome Centre for Cell Biology is supported by core funding from the Wellcome Trust (203149). AS is supported by Asthma Lung UK. We are grateful to Tenovus Tayside for grants to develop our air liquid interface culture system. The UK National PCD Centres are commissioned and funded by NHS England. Study authors and data contributors participate in the BEAT-PCD and EMBARC clinical research collaborations, supported by the European Respiratory Society. A SSM grant from BEAT-PCD COST action funded AS to conduct work for this project in Oslo, Norway. This research is supported the Chancellerie des Universites de Paris (Legs Poix grant) and by RaDiCo, funded by the French National Research Agency under the specific program Investments for the Future, (Cohort grant agreement ANR-10-COHO-0003). SA, EE, ML acknowledge support from the BEAT-PCD network (COST Action 1407 and European Respiratory Society (ERS) Clinical Research Collaboration). Support for this work to AH was provided by the US NIH grant (Award number 1K08HL150223, as well as the Washington University Childrens Discovery Institute (Award number PD-FR-2021-933). The contents are solely the responsibility of the authors and do not necessarily represent the official views of the National Institute of Health. ### Author Declarations I confirm all relevant ethical guidelines have been followed, and any necessary IRB and/or ethics committee approvals have been obtained. Yes The details of the IRB/oversight body that provided approval or exemption for the research described are given below: Eleven affected individuals from eleven unrelated families and their healthy relatives were included in the study (5 females and 6 males). Genomic DNA was extracted from peripheral blood by standard procedures. Signed and informed consent was obtained from the affected individual as well as relatives through approved protocols. For P1 (UOE), the study was approved by the London-West London & Gene Therapy Advisory Committee Research Ethics Committee (REC number 11/LO/0883), P2 by London-Bloomsbury Research Ethics Committee (REC 08/H0713/82; IRAS ID 103488), P3 by the Institutional Ethics Review Board of the University Muenster (2015-104-f-S), and P4 and P5 by the Institutional Review Board from Institut national de la santé et de la recherche médicale (IRB00003888 - approval no.15-259. Protocols for UNC (P6, P7) human studies were approved by the Institutional Review Board at the University of North Carolina and were performed in compliance with ethical regulations. P8 was recruited for WGS as part of the 100,000 Genomes Project, under approved Research Registry Project RR185 'Study of cilia and ciliopathy genes across the 100,000 GP cohort'. For P9, the study protocols were approved by the Institutional Review Board at Washington University in St. Louis. P10 was recruited under studies approved by the Institutional Review Board for Human Use at the University of Alabama at Birmingham (US), in compliance with ethical regulations. P11 was recruited under the Undiagnosed Disease Network protocol 15-HG-0130 approved by the National Institutes of Health Institutional Review Board. I confirm that all necessary patient/participant consent has been obtained and the appropriate institutional forms have been archived, and that any patient/participant/sample identifiers included were not known to anyone (e.g., hospital staff, patients or participants themselves) outside the research group so cannot be used to identify individuals. Yes I understand that all clinical trials and any other prospective interventional studies must be registered with an ICMJE-approved registry, such as ClinicalTrials.gov. I confirm that any such study reported in the manuscript has been registered and the trial registration ID is provided (note: if posting a prospective study registered retrospectively, please provide a statement in the trial ID field explaining why the study was not registered in advance). Yes I have followed all appropriate research reporting guidelines and uploaded the relevant EQUATOR Network research reporting checklist(s) and other pertinent material as supplementary files, if applicable. Yes All data produced in the present work are contained in the manuscript, with details of datasets listed in the Methods section. Total proteomic data are available via ProteomeXchange with identifier PXD036304. Total RNASeq data has been deposited in GEO under accession code GSE214070. ,