# A study into rare GPR146 gene variants in humans and mice

Boyan Zhang, Antoine Rimbert, Antoine Lainé, Nicolette Huijkman, Niels Kloosterhuis, Marieke Smit, Bart van de Sluis, Jan Albert Kuivenhoven, Umesh Tharehalli

► To cite this version:

Boyan Zhang, Antoine Rimbert, Antoine Lainé, Nicolette Huijkman, Niels Kloosterhuis, et al. A study into rare GPR146 gene variants in humans and mice. Atherosclerosis, 2025, 403, 10.1016/j.atherosclerosis.2025.119137. hal-05361359

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Submitted on 12 Nov 2025

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# A study into rare *GPR146* gene variants in humans and mice

<figure><img src="image_2.png" /></figure>

Boyan Zhang<sup>a ID</sup>, Antoine Rimbert<sup>b</sup>, Antoine Lainé<sup>b</sup>, Nicolette Huijkman<sup>a</sup>, Niels Kloosterhuis<sup>a</sup>, Marieke Smit<sup>a</sup>, Bart van de Sluis<sup>a</sup>, Jan Albert Kuivenhoven<sup>a,*,1</sup>, Umesh Tharehalli<sup>a,**,1 ID</sup>

## ARTICLE INFO

**Keywords:**

GPR146
Rare variant
Cholesterol
Mutation

## ABSTRACT

**Background and aims:** G-protein coupled receptor 146 (GPR146)-deficient mice exhibit a moderate 21 % reduction in plasma cholesterol. This is associated with decreased phosphorylation of ERK1/2 and reduced SREBP2 activity in the liver, which leads to lower VLDL secretion. Insight into the role of GPR146 in humans is however limited. We therefore set out to study rare genetic variants in GPR146 to improve our understanding of this new player in lipid metabolism.

**Methods:** We used whole genome sequencing data from UK Biobank participants to search for rare coding variants in *GPR146*. We first carried out gene-based burden tests (using SAIGE-GENE-framework) and examined the association of individual variants with plasma cholesterol levels. One of the variants (P62L) was also studied using the Global Lipids Genetics Consortium (GLGC) data set and in a knock-in mouse model.

**Results:** We found that the combination of rare genetic variants identified in *GPR146* is significantly associated with plasma cholesterol levels. Three rare variants, i.e. P62L, I129I, and A175T were individually associated with reduced plasma cholesterol. In the GLGC cohort, the P62L variant was associated with reductions in both HDL and LDL cholesterol. Follow-up experiments show lower plasma cholesterol levels in *GPR146*<sup>P61L</sup> male and female mice (–13 %, p < 0.05 and –15 %, p < 0.005, respectively) when compared to controls due to a reduction in HDL cholesterol. The *GPR146*<sup>P61L</sup> mice did not exhibit a change in VLDL secretion. In line, the ERK1/2 signalling pathway and *Srebp2* mRNA expression in liver homogenates, and the secretion of apoB by primary hepatocytes of *GPR146*<sup>P61L</sup> and wild-type mice were unchanged.

**Conclusions:** This study shows that rare *GPR146* gene variants are associated with lower plasma cholesterol levels in humans. One of these variants, P62L is associated with reductions of HDL cholesterol and LDL cholesterol in humans while the ortholog in mice confers a loss of *GPR146* function leading to only reduced HDL cholesterol. How *GPR146* affects HDL metabolism in humans and mice remains to be resolved.

## 1. Introduction

In 2013, a variant (rs1997243-G) in the *GPR146* gene locus encoding for G-protein coupled receptor 146 (*GPR146*) was found to be associated with increased plasma cholesterol levels in humans [1]. Later epidemiological studies showed that this variant is associated with increased high-density lipoprotein (HDL) cholesterol as well as low-density lipoprotein (LDL) cholesterol [2,3]. Whole-body as well as liver-specific *GPR146* knockout mice were subsequently shown to exhibit reductions in LDL and HDL cholesterol. Insightful mechanistic studies revealed that *GPR146* affects apoB-containing lipoproteins in plasma via modulation

of very low-density lipoprotein (VLDL) secretion through the ERK1/2-SREBP2 axis [4]. Importantly, *GPR146* deficiency in mice protects against hypercholesterolemia [4,5] and atherosclerosis [4], which has sparked enthusiasm to target *GPR146* with antagonists [6,7]. Since the anti-atherogenic effects are independent of the LDL receptor, *GPR146* antagonists may be of specific benefit to patients suffering from homozygous familial hypercholesterolemia [4]. Recently, a novel gut-derived hormone, named cholesin, was shown to antagonize *GPR146* signalling in the liver [8].

Although the mechanism by which *GPR146* controls plasma cholesterol levels has been studied extensively in mice [4,5], insight into

<footnote><sup>a</sup> Department of Pediatrics, University Medical Center Groningen, University of Groningen, Groningen, the Netherlands</footnote>

<footnote><sup>b</sup> Nantes Université, CNRS, Inserm, Institut du Thorax, F-44000, Nantes, France</footnote>

<footnote>* Corresponding author.</footnote>

<footnote>** Corresponding author.</footnote>

<footnote>E-mail addresses: j.a.kuivenhoven@umcg.nl (J.A. Kuivenhoven), u.tharehalli.mathada@umcg.nl (U. Tharehalli).</footnote>

<footnote n="1">¹ Equal contribution.</footnote>
the role of *GPR146* in human lipid metabolism is primarily based on studies of common *GPR146* gene variants. We have previously shown that increased *GPR146* gene expression in carriers of the common rs1997243-G variant is associated with increased total, LDL and HDL cholesterol. Conversely, carriers of another common *GPR146* variant (rs2362529-C), associated with decreased *GPR146* gene expression, present with decreased levels of total, LDL and HDL cholesterol [9].

To increase insight into the role of *GPR146* in human lipid metabolism, we set out to identify and study rare, possibly high-impact variants in the *GPR146* gene in population-based cohorts. In this effort, we identified three rare variants that are all associated with reduced plasma cholesterol. Using an orthologous whole-body knock-in mouse model, we show that P62L is functional. In mice, it reduces HDL cholesterol but without changing VLDL secretion and plasma LDL cholesterol. Human carriers of this mutation on the other hand show reductions in both HDL and LDL cholesterol leaving the question how *GPR146* affects HDL metabolism in both species.

## 2. Methods

### 2.1. Screening for rare genetic coding variants in *GPR146* in the UK Biobank

The UK Biobank study, described in detail previously, is a population-based prospective cohort in the United Kingdom which included over 500,000 individuals aged between 40 and 69 years from 2006 to 2010 [10]. The study received approval from the Northwest Multi-Centre Research Ethics Committee, the National Information Governance Board for Health and Social Care for England and Wales, and the Community Health Index Advisory Group for Scotland (details available: https://www.ukbiobank.ac.uk/media/0xsbmfmw/egf.pdf). All participants provided informed consent (https://www.ukbiobank.ac.uk/media/gnkeyh2q/study-rationale.pdf). Blood biomarker measurement details are extensively described in the UK Biobank showcase (https://biobank.ndph.ox.ac.uk/showcase/label.cgi?id=17518). Cholesterol plasma levels were measured using enzymatic assays (https://biobank.ndph.ox.ac.uk/showcase/showcase/docs/serum_biochemistry.pdf). This research was conducted using the UK Biobank resource under application number 49823. Records of 120 individuals who withdrew from the UK Biobank were excluded from the analyses.

### 2.2. Rare coding variants in *GPR146*: whole genome sequencing and association tests

We utilized the mixed-model framework SAIGE-GENE to perform group tests [11], including gene-based burden (mean), SKAT (variance), SKAT-O (hybrid variance/mean), and single-variant tests [12]. Our analysis focuses on coding variants with a minor allele frequency below 0.01 and their association with plasma cholesterol levels, normalized for age, sex, and the 10 principal components. We extracted the genotype of variants located in the coding region of *GPR146* from pVCF files of 179, 148 UK Biobank participants using plink2.0 [13]. Variants were annotated using ANNOVAR and categorized as loss-of-function, missense, or synonymous [14]. The analyses were conducted on the UK Biobank platform (https://ukbiobank.dnanexus.com).

To validate the outcomes in participants of the UK Biobank study, we used The Global Lipids Genetics Consortium, aggregated GWAS, results from 1,654,960 individuals across 201 primary studies representing five genetic ancestry groups. Summary statistics from the meta-analysis were downloaded via http://csg.sph.umich.edu/willer/public/glgc-lipids2021.

The linkage disequilibrium between rs151124717 (P62L) and rs2362529 was analysed using the LDpair tool (https://ldlink.nih.gov/?tab=ldpair).

### 2.3. A whole-body *GPR146* p.Proline-61-leucine (*GPR146*<sup>P61L</sup>) knock-in mouse model

A whole-body knock-in *GPR146*<sup>P61L</sup> mouse model was established using CRISPR-Cas9 technology. The sgRNA was designed and selected using the web tool CRISPOR.org [15]. The selected sgRNA target sequence, 5'-TGCCCGACGTGTACTTCGTG-3', is a complementary strand. To produce sgRNA in *vitro* transcription reaction (IVT) was performed using Megascript T7 transcription kit (Thermo Scientific, AM1334) according to manufacturer's instructions, with string DNA (ordered at Thermo Scientific) as a template. The sequences of IVT and repair template are in the Supplementary Table 1. In addition to the P61L variant, two additional synonymous variants (c.C177G and c.G189C) were designed in the repair template to prevent repeated cleaving by Cas9. Bioinformatic analysis, including predictions of RNA splicing sites, miRNA binding sites, mRNA structure, and pathogenicity, predicted no significant impact of these additional variants [16] (Supplementary Figs. 1–2 and Supplementary Tables 2–3). Genomic DNA sequencing of homozygous knock-in mice later confirmed the introduction P61L, and c.C177G but not c.G189C (Supplementary Fig. 3). The qPCR analysis, specifically amplifying mutated and non-mutated regions, showed that *Gpr146* mRNA was not downregulated by the introduction of P61L mutation along with the c.C177G variant (Supplementary Fig. 4).

The plasmid containing spCas9 sequence (pSpCas9(BB)-2A-puro, Addgene 48139), cloned behind T7 RNA promoter, was used as a template for the IVT reaction to generate spCas9 mRNA using T7 Transcription Kit (Thermo, AM1344) according to the manufacturer's instructions. To prepare the injection mix for fertilized eggs, 100 ng/µl spCas9, 50 ng/µl sgRNA, and 100 ng/µl repair template were combined. The mutant mice were generated by injecting this mix into the nucleus of the zygotes collected from pregnant mice with a C57BL/6J genetic background [17]. Pronuclear injected eggs were then transferred via the infundibulum into the oviductal ampulla of pseudopregnant B6CBAF1/J females. The pups were genotyped by polymerase chain reaction (PCR) after birth using genomic DNA extracted from ear tissue. The sequences of the primers used for genotyping are given provided in Supplementary Table 4.

For the experiments, homozygous male and female mice and their wild-type littermates were single-housed with 12-h light/dark cycle and fed a chow diet. Food and water were available ad libitum. All experiments were approved by the Institutional Animal Care and Use Committee, University of Groningen under the study number 2115553-01-005 (Groningen, Netherlands). Prior to orbital bleeding and sacrifice, the mice were fasted for 4 h. The mice were sacrificed by cardiac puncture under anesthesia, and blood and liver tissue were harvested. Blood was collected in EDTA tubes and centrifuged at 3000 rpm for 10 min at 4 °C to separate plasma from blood cells, and liver tissue was snap frozen. All samples were stored at -80 °C until analysis.

### 2.4. Lipid measurements

Total cholesterol levels were measured using colorimetric assays with cholesterol enzymatic reagents (Cholesterol FS, DiaSys #113000010026) and the Cholesterol standard FS (DiaSys, #113003010030) as a reference. Triglyceride levels were assessed using Triglycerides FS (DiaSys #157100010917), with the Precimat Glycerol standard (Roche, #10166588) as a reference. Cholesterol and triglyceride levels in VLDL, LDL, and HDL were analysed through fast protein liquid chromatography (FPLC) with post-column enzymatic reactions, as previously described [18]. To quantify the plasma HDL cholesterol levels, we used PEG6000 to precipitate apoB-containing lipoproteins. PEG6000 was dissolved in 200 mM glycine solution (20 % w/v, pH = 10). In brief, the mix of plasma sample, phosphate-buffered saline (PBS), and PEG6000 solution (3:1:2) was centrifuged at 6000 rpm for 20 min at room temperature. Subsequently, 25 µl supernatant was mixed with 20
µl PEG6000 solution then centrifuged as described above. Eventually, 35 µl supernatant was collected for a standard lipid calorimetric assay as described above. Hepatic lipid extraction and liver histology were performed according to previously established methods [19].

## 2.5. VLDL production

To evaluate *in vivo* hepatic VLDL production, mice were fasted for 5 h before receiving an intraperitoneal injection of poloxamer 407 (16758-250G Merck, 100 mg/ml in phosphate-buffered saline), a compound that inhibits lipoprotein lipase. The administered dose of poloxamer 407 was 1 mg per gram of body weight. Blood samples were collected via tail vein bleeding to establish a baseline (pre-injection) and subsequently at 30-, 60-, 120-, 180-, and 240-min post-injection. Plasma was separated by centrifuging at 3000 rpm for 10 min at 4 °C, and plasma triglyceride levels were measured using the method described above.

## 2.6. RNA isolation and gene expression analysis

Total RNA was extracted from frozen liver tissue. Approximately 30 mg of liver tissue was digested in 1 ml of TRIzol reagent (Qiagen) according to the manufacturer's protocol. RNA concentration was measured using a NanoDrop Microvolume Spectrophotometer (ThermoFisher Scientific), and integrity was assessed via agarose gel electrophoresis. For cDNA synthesis, 1 µg of RNA per sample was reverse transcribed using M-MLV Reverse Transcriptase (ThermoFisher Scientific, Ref. 28025021) following the manufacturer's instructions. The resulting cDNA samples were diluted 1:20 then amplified using SYBR Green Master Mix (Roche, Ref. 06402712001). The qPCR primers used are listed in *Supplementary Table 5*.

## 2.7. Protein isolation and western blotting

Frozen liver tissues were homogenized in NP40 buffer containing 0.1 % Nonidet P-40, 0.4 M NaCl, 10 mM Tris-HCl (pH 8.0), and 1 mM EDTA, supplemented with protease and phosphatase inhibitors (Roche, Switzerland). Protein concentrations were determined using the Bradford assay (Bio-Rad, Germany) and adjusted to 1.5 µg/µl by mixing with a sample buffer containing 10 % glycerol, 1 % beta-mercaptoethanol, 1.7 % SDS, 62.4 mM Tris base (pH 6.8), and 0.1 % bromophenol blue. A total of 30 µg of protein per sample was separated by SDS-PAGE at 150 V for 90 min, followed by transfer to a PVDF membrane (Millipore, Germany) via electrophoresis at 300 mA for 120 min. The membranes were blocked with 5 % BSA for 60 min at room temperature, then incubated overnight at 4 °C with the appropriate primary antibody (listed in *Supplementary Table 6*). The following day, membranes were washed three times with Tris-buffered saline with 0.5 % Tween 20 for 5 min each, both before and after a 2-h incubation with the HRP-conjugated secondary antibody (listed in *Supplementary Table 6*) at room temperature. Protein detection was performed using enhanced chemiluminescence, and images were captured with the ChemiDoc XRS + System. Data were analysed using ImageJ software.

## 2.8. Primary hepatocytes isolation and apoB secretion

The perfusion buffer was prepared by diluting 10x stock buffer (NaCl 81.1 g/l, KCl 3.726 g/l, MgCl₂·6H₂O 1.626 g/l, Na₂HPO₄ 2.848 g/l, and KH₂PO₄ 0.544 g/l) to 1x and supplemented with EDTA buffer (NaHCO₃ 2.1 g/l, EDTA 0.745 g/l, glucose 2.969 g/l, L-Lactic acid-Na-Salt 0.846 g/l, and sodium pyruvate 0.022 g/l). The mouse liver perfusion was conducted under anesthesia, with livers perfused at a rate of 6 ml/min for 1 h using perfusion buffer. Post perfusion, the liver was transferred to a Petri dish containing 25 ml of plating medium (DMEM glutamax with 10 % fetal bovine serum (FBS) and 1 % antibiotic-antimycotic) and the hepatocytes were carefully dissociated: The cell suspension was passed through a 70 µm cell strainer and collected in a 50 ml Falcon tube and

centrifuged at 30 g for 4 min at 4 °C to separate epithelial cells (pellet) and non-epithelial cells (floating). The pellet was resuspended in 8 ml culture medium with 13.9 ml Percoll, and 2.1 ml 10x stock buffer described above. The dead cells were separated through centrifugation at 860g for 6 min at 4 °C. The hepatocytes were resuspended in the plating medium and seeded at a density of 3,000,000 cells in collagen-coated 10 cm cell culture dishes, followed by a 4-h incubation for cell attachment.

**ApoB secretion assay:** After overnight culturing of hepatocytes in plating medium, the cells were washed three times with 1x PBS, and then cultured in 7.5 ml of serum-free DMEM and incubated for 6 h to ensure adequate secretion of apoB. The medium from two dishes was pooled and centrifuged at 2000 rpm for 10 min at 4 °C to remove cell debris. The resulting supernatant was transferred to a protein concentrator (Amicon Ultra-15 Centrifugal Filter Units, Ultracel 10K, Merck Millipore Ltd, Ireland) and the medium was concentrated 50-fold by centrifugation at 3000g for 20 min at room temperature.

The protein concentration of the concentrated medium was determined using Bradford assay. To prepare the samples for detecting secreted apoB, the concentrated medium was mixed with the sample buffer described above and boiled at 95 °C for 5 min. Western blotting, as described above in the *Protein isolation and Western blotting* section, was performed using a 5 % SDS gel to detect the presence of secreted ApoB.

## 2.9. Statistical analysis

Statistical analysis was performed using GraphPad Prism software (Version 10.2.3). Parametric tests were applied to data with a normal distribution, while non-parametric tests were used for data that were not normally distributed. Two-way ANOVA with a Tukey correction for multiple comparisons was used to compare groups. Results are displayed as bar graphs with scattered dot plots representing mean ± SD, and XY graphs showing mean ± SD. A p-value of 0.05 was deemed statistically significant for all experiments.

# 3. Results

## 3.1. Rare coding variants in *GPR146* are associated with cholesterol levels

After extracting and selecting rare coding variants in *GPR146* from whole genome sequencing in the UK Biobank, we tested their combined association with plasma cholesterol levels and found that the combination of rare genetic variants in *GPR146* is significantly associated with decreased plasma cholesterol levels (p-burden = 1.7e-04). This association holds when focusing solely on rare missense and loss of function variants (p-burden = 2.7e-04). *Supplementary Table 7* shows the association of all individual variants with plasma cholesterol.

Specifically, the variants *GPR146*-P62L (missense), *GPR146*-I129I (synonymous), *GPR146*-A175T (missense) were individually associated with reduced plasma cholesterol. Access to individual data of participants of the UK Biobank allowed us to study the associations of these rare variants with plasma levels of total cholesterol, LDL cholesterol, HDL cholesterol and triglycerides. *Table 1* shows that carriers of any of these three rare variants exhibit significantly lower total cholesterol, LDL cholesterol levels but not HDL cholesterol compared to non-carriers. The *GPR146*-A175T was moreover associated with reduced triglycerides.

In the Global Lipids Genetics Consortium dataset which encompasses more than a million of participants, and which includes the UK Biobank data set, *GPR146*-P62L was found to be associated with decreased total cholesterol (p = 1.2e-08), LDL cholesterol (p = 1.4e-04) as well as HDL cholesterol (p = 8.8e-06) levels while no association is observed with triglyceride levels (p = 0.08). Of note, this variant is not in linkage disequilibrium with rs2362529-C, a common *GPR146* variant which is
<table><caption>**Table 1**

GPR146 variants associated with plasma cholesterol levels.</caption><thead><tr><th>chrom_pos_ref_alt</th><th>coding</th><th>prot</th><th>nb non_carriers</th><th>nb carriers</th><th>trait</th><th>mean_non_carriers (±se) [mmol/L]</th><th>mean_carriers (±se) [mmol/L]</th><th>pval.</th><th>corr_pval.<sup>a</sup></th></tr></thead><tbody><tr><td>chr7_1057700_C_T</td><td>c. C185T</td><td>p.P62L</td><td>177890</td><td>1181</td><td>Total cholesterol</td><td>5.72 (±0.0027)</td><td>5.61 (±0.0320)</td><td>6.07E-04</td><td>2.47E-04</td></tr><tr><td>chr7_1057700_C_T</td><td>c. C185T</td><td>p.P62L</td><td>177521</td><td>1178</td><td>LDL-cholesterol</td><td>3.57 (±0.0021)</td><td>3.5 (±0.0248)</td><td>5.48E-03</td><td>3.96E-03</td></tr><tr><td>chr7_1057700_C_T</td><td>c. C185T</td><td>p.P62L</td><td>163115</td><td>1078</td><td>HDL-cholesterol</td><td>1.46 (±0.0009)</td><td>1.45 (±0.0114)</td><td>0.256</td><td>0.134</td></tr><tr><td>chr7_1057700_C_T</td><td>c. C185T</td><td>p.P62L</td><td>177700</td><td>1179</td><td>Triglycerides</td><td>1.74 (±0.0024)</td><td>1.70 (±0.0294)</td><td>0.185</td><td>0.201</td></tr><tr><td>chr7_1057902_C_T</td><td>c. C387T</td><td>p.I129I</td><td>179049</td><td>22</td><td>Total cholesterol</td><td>5.72 (±0.0027)</td><td>5.11 (±0.2706)</td><td>3.43E-02</td><td>2.62E-02</td></tr><tr><td>chr7_1057902_C_T</td><td>c. C387T</td><td>p.I129I</td><td>178677</td><td>22</td><td>LDL-cholesterol</td><td>3.57 (±0.0020)</td><td>3.04 (±0.1956)</td><td>1.30E-02</td><td>1.04E-02</td></tr><tr><td>chr7_1057902_C_T</td><td>c. C387T</td><td>p.I129I</td><td>164173</td><td>20</td><td>HDL-cholesterol</td><td>1.46 (±0.0009)</td><td>1.41 (±0.0783)</td><td>0.524</td><td>0.185</td></tr><tr><td>chr7_1057902_C_T</td><td>c. C387T</td><td>p.I129I</td><td>178857</td><td>22</td><td>Triglycerides</td><td>1.74 (±0.0024)</td><td>1.94 (±0.3392)</td><td>0.565</td><td>0.536</td></tr><tr><td>chr7_1058038_G_A</td><td>c. G523A</td><td>p. A175T</td><td>179057</td><td>14</td><td>Total cholesterol</td><td>5.72 (±0.0027)</td><td>5.04 (±0.2864)</td><td>3.40E-02</td><td>3.18E-02</td></tr><tr><td>chr7_1058038_G_A</td><td>c. G523A</td><td>p. A175T</td><td>178686</td><td>13</td><td>LDL-cholesterol</td><td>3.57 (±0.0020)</td><td>3.01 (±0.2081)</td><td>1.86E-02</td><td>2.0E-02</td></tr><tr><td>chr7_1058038_G_A</td><td>c. G523A</td><td>p. A175T</td><td>164179</td><td>14</td><td>HDL-cholesterol</td><td>1.46 (±0.0009)</td><td>1.39 (±0.0928)</td><td>0.485</td><td>0.377</td></tr><tr><td>chr7_1058038_G_A</td><td>c. G523A</td><td>p. A175T</td><td>178865</td><td>14</td><td>Triglycerides</td><td>1.74 (±0.0024)</td><td>1.28 (±0.1336)</td><td>4.31E-03</td><td>7.56E-03</td></tr></tbody></table>

Rare coding variants, defined as those with a minor allele frequency below 0.01, were associated with plasma total, HDL, and LDL cholesterol levels as well as triglycerides in the UK Biobank using whole genome sequencing data (n = 179,148 participants). Genomic positions are presented in the format: chromosome_posi- tion_reference alleleAlternative allele (GRCh38). Coding and protein positions correspond to NM_138445.4 and NP_612454.1, respectively. For each GPR146 variant (p.P62L, p.I129I, and p.A175T), total cholesterol, LDL cholesterol, HDL cholesterol, and triglyceride levels were compared between carriers and non-carriers in the UK Biobank cohorts. Mean plasma lipid/lipoprotein levels and standard errors are reported in mmol/L. 'Pval' represents the statistical comparisons of mean plasma lipid levels between carriers and non-carriers. 'corr_pvalue' denotes the statistical comparisons of mean plasma lipid levels a normalized for age, sex, and the first 10 principal components between carriers and non-carriers.

also associated with reductions in plasma lipids. The two other rare variants could not be studied in the Global Lipids Genetics Consortium due to the absence of sequencing data.

### 3.2. Homozygous GPR146<sup>P61L</sup> mice have reduced plasma lipids

Using CRISPR/Cas9-mediated gene editing, we changed the proline into a leucine residue at the corresponding 61 amino acid (chr5:139,378,381C > T(GRCm39)) of GPR146 in C57BL/6J mice (Supplementary Fig. 3). Homozygous mice were born at the Mendelian 1:2:1 ratio and do not present apparent abnormalities nor changes in body weight gain compared to controls (Supplementary Fig. 5A and B). Using primers designed to amplify both the mutated and non-mutated regions we show that *Gpr146* mRNA expression was not affected in males but increased in female knock-in mice (Supplementary Fig. 4A–F).

On a regular chow diet, male and female GPR146<sup>P61L</sup> mice showed 13 % and 15 % reductions, respectively, in plasma levels of cholesterol compared to wild-type littermates (p < 0.05 and p < 0.005 respectively; Fig. 1A and F). FPLC analysis revealed that this was due to a reduction in HDL cholesterol in both sexes (Fig. 1B and G). Measuring HDL choles- terol following the precipitation of apoB-containing lipoproteins revealed a non-significant 15 % reduction in males and significant reduction in females (16 %, p = 0.005) (Fig. 1C and H; Supplementary Fig. 4I–K). Male GPR146<sup>P61L</sup> mice also exhibited a 21 % reduction in plasma triglycerides (p = 0.03; Fig. 1D) due to a decrease in VLDL tri- glycerides (Fig. 1E), but this effect was not observed in females (Fig. 1I and J). In conclusion, the exchange of Proline to Leucine at position 61 of GPR146 in mice causes reductions in total cholesterol, and HDL cholesterol with sex-specific reduction in plasma triglycerides.

### 3.3. GPR146<sup>P61L</sup> mice do not present with changes in VLDL secretion, ERK1/2 phosphorylation and apoB secretion

Previous studies have shown that the reduction of plasma lipids in GPR146-/- mice is associated with a decrease in VLDL secretion [4,5]. However, male and female GPR146<sup>P61L</sup> mice do not show differences in VLDL secretion compared to control mice, as determined by studying plasma triglyceride levels after poloxamer injection (Fig. 2A and B). In line, we observed no changes in ERK1/2 phosphorylation (Fig. 2C and D), Srebp2 expression (Supplementary Fig. 4G and H) and LDL receptor (LDLR) expression (Supplementary Fig. 4L and M) in liver homogenates of knock-in and control mice. Next, we isolated primary hepatocytes of both groups to study apoB secretion. In line, we observed no differences in the secretion of apoB-100 and apoB-48 (Fig. 2E and F) between the groups. These data together, indicate that the GPR146 P61L variant does not affect VLDL secretion in mice.

### 3.4. GPR146<sup>P61L</sup> mice do not present with changes in hepatic lipid homeostasis

In a previous study, we observed that human GPR146 gene variant carriers not only present with changes in plasma lipid levels but also in plasma liver enzymes [9]. In this light, we investigated the effects of GPR146<sup>P61L</sup> on liver lipid homeostasis. After 35 weeks on a chow diet, livers of GPR146<sup>P61L</sup> and control mice appeared macroscopically normal (Fig. 3A and F). Liver weight and liver-to-body weigh and ratio were unchanged in male GPR146<sup>P61L</sup> mice (Fig. 3B) but slightly increased in females (Fig. 3G). Liver histology, hepatic cholesterol, and triglycerides were unchanged in both sexes (Fig. 3C, D, H, I). Only male GPR146<sup>P61L</sup> mice showed a small significant increase in plasma AST levels (Fig. 3E and J). Taken together, the proline-to-leucine substitution at position 61 in GPR146 of mice has no marked effects on hepatic lipid metabolism and liver function.
<figure><img src="image_3.png" /><figcaption>**Fig. 1.** Impact of the P61L mutation on plasma lipid profiles in mice.</figcaption></figure>

In both male and female *GPR146*<sup>P61L</sup> knock-in mice, plasma cholesterol levels were reduced compared to controls (A, F) which was related to a reduction of HDL cholesterol as assessed by FPLC using pooled plasma sample (B, G) and HDL cholesterol concentration after PEG6000 precipitation (C, H). Male *GPR146*<sup>P61L</sup> mice also showed a 21 % reduction in plasma triglycerides corresponding to a reduction in VLDL triglycerides (D, E) but no changes in triglyceride were observed in the female knock-in mice (I, J). Data Representation: Panels A, C, D, F, H, and I are presented as bar graphs showing mean ± standard deviation (SD). Panels B, E, G and J are depicted as XY plots. Group size and statistical analysis: For panels A, C, D, F, H, and I, the group size was n = 11–12 per group and per gender, with data analysed using a parametric *t*-test. For panels B, E, G, and J, plasma samples were pooled from the respective groups (n = 11–12 per group and per gender). Statistical significance is indicated by asterisks: \*p < 0.05, \*\*p < 0.005; ns indicates not significant.
<figure><img src="image_4.png" /><figcaption>**Fig. 2.** *GPR146* p.P61L mutation does not impact hepatic VLDL secretion

Hepatic VLDL secretion was assessed in both (A) male and (B) female *GPR146*<sup>WT</sup> and *GPR146*<sup>P61L</sup> mice. For both sexes, we did not observe significant differences in triglyceride secretion between knock-in mice and controls. (C) Western blot analysis and (D) relative quantification of ERK phosphorylation and total ERK levels in liver tissues from male *GPR146*<sup>WT</sup> and *GPR146*<sup>P61L</sup> mice. (E) Western blot of apoB-100 and apoB-48 in cell lysates and corresponding media from primary hepatocytes derived from *GPR146*<sup>WT</sup> and *GPR146*<sup>P61L</sup> mice. (F) Quantitative analysis of apoB-100 and apoB-48 levels in the media fraction. Data Representation: Panels A and B are depicted as XY plots with mean ± standard deviation (SD), while panels D and F are presented as bar graphs with mean ± SD. Group size and statistical analysis: For panels A and B, the group size was n = 4–5 for male, n = 6 for female, with data analysed using a Two-Way ANOVA. For panels C and D, n = 6 per group, and for panels E and F, n = 3 per group. Statistical analysis for Panels D and F was performed using a non-parametric *t*-test. Statistical significance is indicated as follows: ns indicates not significant.</figcaption></figure>

## 4. Discussion

Since its discovery, the role of *GPR146* in human lipid metabolism has mostly been carried out using common variants as genetic instruments. Since the effect sizes of such variants are often limited, we here focused on rare variants – which anticipated larger effect size – to improve our understanding of how *GPR146* affects lipids metabolism as previously demonstrated for major lipid genes such as *LDLR*, *PCSK9* and *ANGPTL3* [20–22]. Using a gene-based burden test analysis, we show that the presence of rare variants in *GPR146* is associated with plasma cholesterol levels. In addition, three rare coding variants are significantly associated with lower cholesterol levels, suggesting that they all confer loss of function when considering that *Gpr146*−/− mice have lower plasma cholesterol [4,5]. To validate this assumption, we studied the most significantly associated variant, *GPR146*-Pro62Leu, for an in-depth analysis in a corresponding knock-in mouse model (*GPR146*<sup>P61L</sup>). The rare variant, located in the first cytoplasmic loop of the protein, is predicted to be damaging [9] and was previously shown to co-segregate with low LDL cholesterol in a family [9]. Our results

support that this variant is functional and recapitulates reductions in total cholesterol and HDL cholesterol as observed in humans in this study.

We previously showed that male and female *GPR146*−/− mice have 20 % and 25 % lower plasma cholesterol levels compared to controls, respectively [4]. The current study reveals that male and female *GPR146*<sup>P61L</sup> mice present with 13 % and 15 % reductions in plasma cholesterol compared to controls, respectively. Although we have not carried out a head-to-head comparison, these studies suggest that the P61L mutation in mice has a marked effect on *GPR146* function. The studies in *GPR146* mouse models thus far also suggest that there is sexual dimorphism, but we did not find support for this when studying male and female carriers of the P62L variant in the Global Lipids Genetics Consortium (Supplementary Fig. 6).

In addition to a reduction in plasma cholesterol content, male *GPR146*<sup>P61L</sup> mice also show moderate reductions in plasma triglycerides, again mimicking the effects seen in whole-body and liver-specific *GPR146* knockout mice [4]. Previous studies in whole-body and liver-specific *GPR146* knockout mice have shown that the reductions in
<figure><img src="image_5.png" /><figcaption>Fig. 3. Hepatic characterization of *GPR146<sup>P61L</sup>* mice.</figcaption></figure>

Hepatic characterization of male (A–E) and female (F–J) *GPR146<sup>P61L</sup>* mice. (A, F) Macroscopic images of livers from *GPR146<sup>WT</sup>* and *GPR146<sup>P61L</sup>* mice. (B, G) Liver weight and liver-to-body weight ratios were not significantly different in male *GPR146<sup>P61L</sup>* mice compared to controls. Female *GPR146<sup>P61L</sup>* mice showed a slight increase in liver weight. (C, H) Liver histology with H&E staining showed normal hepatic architecture in *GPR146<sup>P61L</sup>* mice. (D, I) Hepatic cholesterol and triglyceride levels were similar between *GPR146<sup>P61L</sup>* and *GPR146<sup>WT</sup>* groups. (E, J) Plasma AST levels were slightly increased in male *GPR146<sup>P61L</sup>* mice, while other parameters such as ALT and albumin were unaffected. Data Representation: Panels B, D, E, G, I and J are presented as bar graphs showing mean ± standard deviation (SD). Group size and statistical analysis: For panels B, D, E and J, the group size was n = 11–12 per group, with data analysed using a parametric *t*-test. For panels G and I group size was n = 5, with data analysed using a non-parametric *t*-test. Statistical significance is indicated by asterisks: \*p < 0.05; ns indicates not significant.

cholesterol and triglycerides were related to reduced VLDL secretion. With a focus on the reduction in LDL cholesterol, the same investigators show that this is associated with decreased phosphorylation of ERK1/2 and downregulation of SREBP2, a key transcription factor in the *de novo* cholesterol synthesis [4]. In our *GPR146<sup>P61L</sup>* knock-in mice VLDL secretion was unaffected. In line, we did not find evidence for changes in the ERK1/2 and SREBP2 pathway in liver homogenates. In addition, we found no differences in apoB secretion between primary hepatocytes of knock-in and control mice.

Interestingly, the P61L mutation in our mouse model primarily reduces HDL cholesterol levels while whole-body knockout mice show reductions in both HDL and LDL cholesterol [4]. Possibly, the effect of the P61L mutation on LDL cholesterol in mice is too weak to be picked up. On the other hand, our study in human carriers of the orthologous P62L mutation instead shows that the impact on LDL cholesterol is stronger than on HDL cholesterol. This mutation may thus have differential effects in mice and humans.

Other possible downstream consequences of attenuated *GPR146* function may include changes in liver homeostasis. We previously noted that human carriers of *GPR146* gene variants present with changes in circulating liver enzymes [9], but we did not observe overt changes in liver lipid homeostasis and liver enzymes in the *GPR146<sup>P61L</sup>* mice compared to controls.

Although the current study provides evidence that *GPR146* P61L mutation reduces plasma cholesterol, we only marginally studied two other rare variants that are shown to be associated with reduced cholesterol in humans (p.I129I and p.A175T; Table 1). Bioinformatic analysis does not predict a functional impact of p.I129I (Supplementary Figs. 1–2 and Tables 2–3), suggesting that it is in linkage disequilibrium with a functional variant in this gene locus. In-depth studies will also be needed to study the functionality of the p.A175T variant, which is

predicted to have a functional effect, especially since it appears to affect triglyceride levels (Supplemental Table 3). The current absence of *in vitro* methods to assess effects on *GPR146* function would, however, again require the generation of an animal model.

In conclusion, this study shows that rare variants in the *GPR146* gene are associated with plasma cholesterol levels in humans (Summarised in Fig. 4). The study also reveals the first genetic evidence that the rare human P62L variant is a loss-of-function mutation that confers a reduction in plasma levels of total cholesterol, and HDL cholesterol in *GPR146<sup>P61L</sup>* mice. The moderate reductions in plasma lipids in these mice cannot be explained by changes in hepatic VLDL and hepatocyte apoB secretion. The current and previous studies show that *GPR146* affects HDL cholesterol levels consistently. Possibly, *GPR146* regulates plasma levels of HDL cholesterol level by reducing hepatic HDL production. Alternatively, it may regulate hepatic uptake of HDL cholesterol thereby increasing reverse cholesterol transport. The overall modest effect of *GPR146* deficiency on plasma lipids and the even more moderate effects of the P61L mutation in mice, however, make further studies challenging.

## CRediT authorship contribution statement

**Boyan Zhang:** conducted experimental mouse and bioinformatic studies, Writing – original draft. **Antoine Rimbert:** helped to design the study and, Writing – original draft. **Antoine Lainé:** conducted the genetic, Formal analysis, in human cohorts. **Nicolette Huijkman:** design and generation constructs to make a knock-in mouse model. **Niels Kloosterhuis:** generation of knock-in mouse model. **Marieke Smit:** generation of knock-in mouse model. **Bart van de Sluis:** mentoring the making and characterization of the mouse model and critical input on the manuscript. **Jan Albert Kuivenhoven:** Conceptualization, of overall
Functional validation of rare GPR146 gene variant

<figure><img src="image_6.png" /><figcaption>**Fig. 4.** Graphical summary
This study highlights that rare coding variants in the *GPR146* gene are associated with decreased plasma total cholesterol levels in humans. Among these, the most frequent variant, P62L, is shown to affect *GPR146* function. In an orthologous knock-in mouse model (GPR146<sup>P61L</sup>), this mutation causes moderate reductions in total and HDL cholesterol but not LDL cholesterol.</figcaption></figure>

study design, mentoring PhD student and post-doctoral fellow, Writing –
original draft, realization of, Funding acquisition, of the project. Umesh
Tharehalli: Conceptualization, of overall study design, conducted
experimental mouse studies and was responsible for writing the
manuscript.

Declaration of interest

The authors declare that they have no known competing financial
interests or personal relationships that could have appeared to influence
the work reported in this article.

Acknowledgments

Boyan Zhang is supported by a joint fellowship from the China Scholarship Council and University Medical Center Groningen (CSC202006170047). Antoine Rimbert is supported by the GENESIS project funded by the Agence Nationale de la Recherche (ANR-21-CE14-0051). Jan Albert Kuivenhoven and Bart van de Sluis are supported by grants from ZonMW (09120012110057) and European Union (MSCA-ITN-2020, 953489), Bart van de Sluis from ENW NWO (OCENW. M.22.034). Umesh Tharehalli is supported by the European Atherosclerosis Society (EAS) Competitive Grants for Young Researchers 2023 (760-2023-03). The present research has been conducted using the UK Biobank resource under the application number 49823. We are most grateful to the Bioinformatics Core Facility of Nantes BiRD, member of Biogenouest, Institut Français de Bioinformatique (IFB) (ANR-11-INBS-0013) for the use of its resources and for its technical support.

**Appendix A. Supplementary data**

Supplementary data to this article can be found online at https://doi.org/10.1016/j.atherosclerosis.2025.119137.

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