Small-molecule functional rescue of PIEZO1 channel variants associated with generalised lymphatic dysplasia

Generalised Lymphatic Dysplasia (GLD) is characterised by widespread lymphoedema, with at least one of the following: fetal hydrops, intestinal or pulmonary lymphangiectasia, pleural effusions, pericardial effusions and ascites. Satisfactory medical therapies are lacking. A genetic association has been identified that prevents expression or surface trafficking of PIEZO1, a subunit of mechanically activated calcium-permeable channels. However, PIEZO1 is a large and highly polymorphic gene and interpretation of variants identified in this gene can be challenging. PIEZO1- related GLD with non-immune fetal hydrops is autosomal recessive, however, heterozygous variants in PIEZO1 (often gain-of-function) causing Dehydrated Hereditary Stomatocytosis (DHS) (a relative mild anaemia), may also present with perinatal non-immune hydrops (not caused by anaemia). Here we sought to develop methods to confirm pathogenicity of missense variants of uncertain significance in PIEZO1 , to gain deeper understanding and pharmacological solutions. Four novel GLD-associated missense variants in PIEZO1 are identified that express and surface localise as full-length protein but with reduced or abolished mechanically activated channel function. Yoda1, a small-molecule agonist, functionally rescues the channels and their physiological regulation by mechanical force and hypo-osmolality. The GLD-associated variants mediate intracellular calcium release as well as calcium entry, suggesting two pools of channels and opportunity for increased rescue through access to the intracellular pool. New Yoda1 analogues are also identified that improve rescue. The functional assays have assisted the interpretation of the variants of uncertain significance as the data suggest loss of PIEZO1 force sensing as a cause of the GLD observed in the patients. The potential to pharmacologically overcome the loss of force sensing was demonstrated and supports the concept of stimulation of PIEZO1 with an agonist to address wide-ranging problems of lymphatic insufficiency. ![Figure][1] HIGHLIGHTS 1. Previously unrecognised variants in PIEZO1 that associate with GLD are identified and characterised and pathogenicity confirmed 2. The variants encode single amino acid changes that inhibit PIEZO1 channel activation by physiological mechanical forces 3. A small-molecule agonist rescues the channels and their physiological regulation 4. Variants are partly intracellular, suggesting an opportunity for improved rescue through the use of intracellular-acting agonists 5. New agonists are identified that improve rescue, suggesting routes to medical therapies for GLD and potentially other disorders of lymphatic insufficiency ### Competing Interest Statement D.J.B. and R.F. are partners of CalTIC GmbH, a pharmaceutical startup company with a mission to develop ion channel modulators as new classes of medicines. No other conflicts of interests are disclosed. ### Funding Statement The work was supported by research grants from Wellcome (grant number 110044/Z/15/Z) and British Heart Foundation (BHF) (RG/17/11/33042, SP/13/5/30288) and a joint MRC/BHF program grant (MR/P011543/1 and RG/17/7/33217) and Newlife Foundation for Disabled Children (12-13/01). Studentships from University of Leeds (for K.C.) and the BHF (FS/4yPhD/F/20/34130 for K.A.S. and FS/18/78/33932 for E.D.). For the purpose of Open Access, the authors have applied a CC BY public copyright license to any Author Accepted Manuscript version arising from this submission. ### 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: Ethical approval for this study was obtained from the South West London Research Ethics Committee (REC Ref: 05/Q0803/257) and written informed consent was obtained from all participants. Permission to publish was also obtained. 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, such as any relevant EQUATOR Network research reporting checklist(s) and other pertinent material, if applicable. Yes The data that support the findings of this study are available from the corresponding author upon reasonable request. Some data may not be made available because of privacy or ethical restrictions. All original data are available in an Excel file. Unique laboratory materials created in the project are available on request (D.J.B. for biological materials and R.F. for chemicals). [1]: pending:yes