Large-scale functional characterization of low-density lipoprotein receptor gene variants improves risk assessment in cardiovascular disease

Aims Lack of functional information for low-density lipoprotein (LDL) receptor (LDLR) mutations limits the use of genetics for early diagnosis, risk assessment and clinical decision making in familial hypercholesterolemia (FH). The goal of this study was an in-depth and large-scale functional characterization of LDLR variants to overcome this problem. Methods Open-source robotic tools were integrated with multiplexed high-content microscopy, image and data analysis into a novel semi-automated analysis pipeline for the characterization of LDLR variants to quantify LDL uptake, LDLR localization and expression. Results 315 LDLR coding variants were functionally characterized in this study and collapsed into four functional groups based on their residual LDL uptake activity (“Loss-of-function”, 0-10% activity; “defective”, 10-30%; “mildly-defective”, 30-70%; and “non-defective”, > 90%). Integration of the activity groups with whole-exome sequencing and clinical data from UK biobank demonstrated that considering LDLR activity levels improved risk assessment in dyslipidaemia and cardiovascular disease (CVD). Individuals carrying LDLR variants from the loss-of-function and defective groups displayed increased odds ratios for CVD (OR=6.1, 95% CI = 1.5 - 24.4; OR = 1.83, 95% CI = 1.2 - 2.7) as compared to the non-defective group. Also, plasma LDL-cholesterol, utilization of lipid-lowering drugs and combination therapy were higher in the loss-of-function (OR = 15.4, 95% CI = 3.8 - 61.7; OR = 7.6, 95% CI = 1.8 - 31.8; OR = 96.8, 95% CI = 22.6 - 414.1), defective (OR = 5.9, 95% CI = 4.1 - 8.6; OR = 3.5, 95% CI = 2.5 - 4.9; OR = 15.6, 95% CI = 8.4 - 29.1) and mildly-defective group (OR = 2.0, 95% CI = 1.5 - 2.7; OR = 2.0, 95% CI = 1.6 - 2.4; OR = 1.9, 95% CI = 1.0 - 3.4) as compared to the non-defective group. Especially, the loss-of-function group displayed higher CVD risk, increased LDL-C and combination therapy usage as compared to the ClinVar pathogenic group for the same subjects. Furthermore, the functional data indicates that prediction tools tend to overestimate the fraction of pathogenic LDLR variants. Conclusion Systematic functional data for LDLR variants paves the way for improved diagnosis, risk assessment and treatment optimization for FH patients, enabling a better utilization of genetic data in clinical decision making. Translational perspective A loss-of-function LDLR variant leads to lifelong exposure of elevated LDL-C. Whilst sequencing of the LDLR gene is included in the genetic assessment of FH patients, most LDLR variants lack information about functional consequences at the cellular level. This limits the utility of genetic tools in the diagnosis and treatment of FH. This study overcomes this problem, providing functional information for a large set of LDLR variants. Integration with genetic and clinical data from UK biobank enables links between functional and clinical effects, making it easier to diagnose FH and estimate a patient’s cardiovascular risk. ### Competing Interest Statement The authors have declared no competing interest. ### Funding Statement Grants from the Academy of Finland 328861 and 325040, Business Finland (Research to Business) 1821/31/2021, Magnus Ehrnrooth and the Foundation for Cardiovascular Research to S.G.P. Grants from Academy of Finland Center of Excellence in Complex Disease Genetics 312062, Academy of Finland 285380, the Finnish Foundation for Cardiovascular Research and the Sigrid Juselius Foundation to S.R. Funding from the Doctoral Programme in Population Health, University of Helsinki to M.T. ### 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: This project has been granted access to use the UK Biobank resource. Ethical application has been granted to UK Biobank. Application Number: 87446 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 All data produced in the present study are available upon reasonable request to the authors