The functional landscape of coding variation in the familial hypercholesterolemia gene LDLR

Abstract

Variants in the familial hypercholesterolemia gene LDLR-the most important genetic driver of cardiovascular disease-can raise circulating low-density lipoprotein (LDL) cholesterol concentrations and increase the risk of premature atherosclerosis. Definitive classifications are lacking for nearly half of clinically encountered LDLR missense variants, limiting interventions that reduce disease burden. We tested the impact of ~17,000 (nearly all possible) LDLR coding variants on both LDLR cell-surface abundance and LDL uptake, yielding sequence-function maps that recapitulate known biochemistry, offer functional insights, and provide evidence for interpreting clinical variants. Functional scores correlated with hyperlipidemia phenotypes in prospective human cohorts and augmented polygenic scores to improve risk inference, highlighting the potential of this resource to accelerate familial hypercholesterolemia diagnosis and improve patient outcomes.

Fig. 1.. A sequence–function map of the familial hypercholesterolemia gene LDLR.

(A) Overview of the experimental workflow and (B) phenotypic selections used in this study. (C) Functional scores measuring LDL uptake for all possible amino acid substitutions in LDLR. Color scale is shown at the bottom right: damaging (blue); normal (white); above normal (red); unmeasured (gray); reference amino acid (yellow). The vertical axis represents each possible amino acid (one-letter code) grouped by hydrophobic (hyd.) and polar residues (pol.; +/−). Error bars indicate standard error. (D) Score distributions for synonymous (green), missense (gray), and nonsense (blue) variants. (E) Functional scores as a measure of log (minor allele frequency) for variants present in the UK Biobank (dark gray), All of Us (light gray), and both cohorts (white).MAF, minor allele frequency; Hyd, hydrophobic; Pol, polar; UKB, UK Biobank; AoU, All of Us.

Fig. 2.. Functional scores capture atomic-level details of the LDLR-ApoB100 interaction.

(A) Ribbon diagram of LDLR (LA2 to EGF-A) bound to ApoB100 (residues 1833–3892; yellow) (PDB 9BDE). LDLR is colored by functional score (residue mean) scaled as in Fig. 1C. Insets show (B) LA5, (C) LA6, and (D) LA7-EGF-A (partial). ApoB100 LDLR binding sites A and B are shown in cyan; Ca²⁺ ions are shown in gray; FH variants in ApoB100 are annotated in red.

Fig. 3.. Mapping variant function across readouts highlights functional patterns in the ligand-binding domain.

Functional scores for all possible amino acid substitutions in the LDLR ligand-binding domain (LA modules 1–7) measuring (top) LDLR cell-surface abundance; (middle) LDL uptake measured without VLDL (− VLDL); and (bottom) LDL uptake with a 4:1 stoichiometric excess of VLDL (+ VLDL). Functional scores are scaled as in Fig. 1C. Conserved disulfide-forming residues (:) and acidic Ca²⁺-coordinating (*) residues are annotated. Error bars indicate standard error. VLDL, very low-density lipoprotein.

Fig. 4.. Functional scores enable HeFH risk quantification and diagnosis.

(A) Risk (odds ratio) of “probable” familial hypercholesterolemia (HeFH) for UK Biobank participants who are carrying LDLR variants of a given type: nonsynonymous variants (top); ClinVar variants lacking a definitive classification (VUS) (middle); or pathogenic (P/LP) variants (bottom). In each, risk is relative to noncarriers and is stratified by functional score. Dotted line denotes the odds ratio of HeFH among all LDLR variant carriers. (B) Receiver operating characteristic (ROC) curve describing the ability of functional scores to distinguish between two groups of LDLR variant carriers in the UK Biobank: those defined as having “definite” HeFH under Dutch Lipid Clinic Network criteria; and those not suspected of having HeFH, without considering genotype for either group (AUROC = 0.87.).

Fig. 5.. Refining risk estimation for HeFH-related phenotypes.

(A) The correlation of functional scores with the frequency of elevated LDL-C (LDL-C ≥ 190 mg/dL) (Spearman’s ρ = −0.49; P = 0.01) among UK Biobank participants. The probability of each phenotype was assessed within a given functional score window (0.05 +/− 0.025) with > 5 participants per window. The dotted line denotes the mean of this measure among all LDLR variant carriers. (B) Elevation in LDL-C (mg/dL) among participants in the UK Biobank carrying an LDLR variant grouped by functional score; elevation is shown relative to the UK Biobank population average (146 mg/dL). Error bars denote standard error. The adjusted odds ratio of (C) hyperlipidemia (LDL-C ≥ 190 mg/dL) for participants stratified by functional score [normal (gray) and damaging (black)] and polygenic risk score quartile (for LDL-C and CAD, respectively). Odds ratios are adjusted for age, sex, BMI, and ethnicity. Non-carriers with intermediate PRS are used as reference. The dotted line denotes an OR of 1. (D) Time-to-hyperlipidemia diagnosis for UK Biobank participants—stratified by functional score, PRS, and both—is shown for participants with a: 25^th percentile PRS (dotted gray); 75^th percentile PRS (dotted pink); damaging functional score (solid pink); or 75^th percentile PRS and damaging functional score (solid red). Time-to-diagnosis among all participants is shown as reference (dotted black). (E and F) Same as (C and D) assessing the odds ratio coronary artery disease (CAD) and CAD-free survival, respectively. Error bars in (C to F) indicate the 95^th percentile CI. LDL, low-density lipoprotein; OR, odds ratio; HeFH, familial hypercholesterolemia; CAD, coronary artery disease; PRS, polygenic risk score; Norm., normal; Dam., damaging.