The WWOX Gene Modulates High-Density Lipoprotein and Lipid MetabolismClinical Perspective
Background—Low levels of high-density lipoprotein (HDL) cholesterol constitutes a major risk factor for atherosclerosis. Recent studies from our group reported a genetic association between the WW domain-containing oxidoreductase (WWOX) gene and HDL cholesterol levels. Here, through next-generation resequencing, in vivo functional studies and gene microarray analyses, we investigated the role of WWOX in HDL and lipid metabolism.
Methods and Results—Using next-generation resequencing of the WWOX region, we first identified 8 variants significantly associated and perfectly segregating with the low-HDL trait in 2 multigenerational French Canadian dyslipidemic families. To understand in vivo functions of WWOX, we used liver-specific Wwoxhep−/− and total Wwox−/− mice models, where we found decreased ApoA-I and Abca1 levels in hepatic tissues. Analyses of lipoprotein profiles in Wwox−/−, but not Wwoxhep−/− littermates, also showed marked reductions in serum HDL cholesterol concentrations, concordant with the low-HDL findings observed in families. We next obtained evidence of a sex-specific effect in female Wwoxhep−/− mice, where microarray analyses revealed an increase in plasma triglycerides and altered lipid metabolic pathways. We further identified a significant reduction in ApoA-I and Lpl and an upregulation in Fas, Angptl4, and Lipg, suggesting that the effects of Wwox involve multiple pathways, including cholesterol homeostasis, ApoA-I/ABCA1 pathway, and fatty acid biosynthesis/triglyceride metabolism.
Conclusions—Our data indicate that WWOX disruption alters HDL and lipoprotein metabolism through several mechanisms and may account for the low-HDL phenotype observed in families expressing the WWOX variants. These findings thus describe a novel gene involved in cellular lipid homeostasis, which effects may impact atherosclerotic disease development.
Low plasma high-density lipoprotein cholesterol (HDL-C) constitutes a major and coherent cardiovascular risk factor.1 HDL-C levels are regulated by a combination of environmental and genetic factors, with heritability estimates of 40% to 60%,2 emphasizing the need to characterize novel genetic regulators of HDL metabolism.
Clinical Perspective on p 504
We previously identified a region-wide significant association between low HDL-C and the WW domain-containing oxidoreductase (WWOX) gene on chromosome 16q23-24, in dyslipidemic families and low HDL-C cases and controls.3 Genome-wide association studies4,5 have also shown that WWOX is strongly associated with HDL-C, triglyceride (TG) levels,6 and left ventricular thickness.7 Its specific role in cellular lipid homeostasis and lipoprotein metabolism remains however unknown.
The WWOX gene spans 1.1 Mb at the common fragile site FRA16D (chr16q23).8,9 It encodes a 46-kDa tumor suppressor,10,11 the expression of which is altered in several types of human malignancies.10–12 Wwox disruption in mice results in metabolic abnormalities, impaired growth, and postnatal lethality, implying an indispensable role for Wwox in metabolism.10,13 Its interactions are believed to be largely driven by binding to proline-rich PPxY motifs found within an array of potential ligands, such as p73, RUNX, c-Jun, AP2, and NF-κB transcription factors, as well as several other cellular proteins including SIMPLE, ErbB4, and Ezrin.14–18 Furthermore, WWOX is expressed within various tissues, regulating a wide variety of cellular functions such as protein degradation, transcription, cellular trafficking, and metabolic reactions.19 The highest WWOX expression was detected in hormonally regulated tissues (testis, ovary, prostate, and liver). This expression pattern, coupled with the presence of a short-chain dehydrogenase domain, suggests a role for WWOX in steroid metabolism. Moreover, it was recently observed that Wwox knockout (KO) mice exhibit marked reductions in serum lipid levels and display impaired gene expression of key stereoidogenic enzymes.10,20
We therefore sought to characterize WWOX as a novel genetic determinant involved in HDL-C regulation. Using a combination of next-generation resequencing in HDL-deficient families, in vivo functional studies by means of total Wwox KO (Wwox−/−) and Wwox liver-specific KO (Wwoxhep−/−) mouse models, along with conventional and modern gene microarray analyses, we demonstrate the role of WWOX in HDL and lipid metabolism.
Materials and Methods
Mice were maintained in a clean, modified-barrier animal facility. Animals were fed a standard rodent chow diet (Harlan Laboratory, Indianapolis, IN) and water ad libitum, unless otherwise mentioned, under controlled light (12 L:12D) and temperature (68–74°F). Animal research was approved by the University of Texas M. D. Anderson Cancer Center Institutional Animal Care and Use Committee (Animal Welfare Assurance Number A3343-01), the Hebrew University-Hadassah Medical School, and the McGill University Animal Care Committee. Procedures followed were in accordance with institutional guidelines.
Generation of Total Wwox-Deficient and Wwox liver-specific KO Mice
Total Wwox KO (Wwox−/−) mice were generated as previously described.10 Wwox liver-specific KO mice (Wwoxhep−/−) were produced by crossing female Wwoxflox/flox (129SV/C57Bl/6 background, previously generated)11 with male Alb-Cre transgenic mice having the Cre-recombinase gene under the control of the Albumin promoter (pure C57Bl6/J inbred from Jackson Laboratories).21 The Wwoxflox/WT;AlbCre+/− mice were intercrossed to obtain Wwoxflox/flox;AlbCre+/− mice (Wwoxhep−/−). Genotypes were determined by polymerase chain reaction (PCR) using the oligonucleotide primers: Cre F: 5′-GCCTGCATTACCGGTCGATGCAACG-3′; Cre R: 5′-GTGGCAGATGGCGCGGCAACACCAT-3′; Wwox-N1: 5′-ATGGGCCGAAACTGGAGCTCAGAA-3′; Wwox-N2: 5′-TCAGCAACTCACTCTGGCTTCAAC-3′; and Wwox-L: 5′-GCATACATTATACGAAGTTATTCGAG-3′.
Lipid Measurements and Lipoprotein Separation Assays
Serum was isolated by centrifugation (3000 rpm, 15 minutes, 4°C) from blood extracted from mice fasted for 4 to 6 hours. For lipid measurements, serum samples (150 μL) were shipped on dry ice to LipoScience Inc (Raleigh, NC) where levels of total cholesterol, HDL-C, low-density lipoprotein cholesterol, TG, ApoA-I, apoB, and glucose were measured using an Olympus AU400e immunoautoanalyzer. Liposcience measurement assays were previously validated in humans, and lipid values falling below human range linearity were excluded from analyses (apoB, ApoA-I, and low-density lipoprotein cholesterol). For lipoprotein profiles separation, 250 μL of serum was separated by high-performance liquid chromatography (HPLC) equipped with a Superose 6 10/300 GL column (GE Healthcare) attached to a Beckman Coulter System Gold apparatus. A 150 mmol/L NaCl mobile phase with a flow rate of 0.4 mL/min was used for separation of samples into 72×400 μL-HPLC fractions that were collected in a 96-well plate using the ProteomeLab automated fraction collector (Beckman Coulter). Cholesterol and TG content in each fraction was determined enzymatically (Infinity kit; Thermo Scientific) according to manufacturer’s instructions. ApoA-I-containing particles were separated by 2-dimensional polyacrylamide gradient gel electrophoresis (2D-PAGGE) as described previously.22 Briefly, serum samples (25 μL) were separated in the first dimension (according to their charge) by 0.75% agarose gel electrophoresis (100 V, 3 hours, 4°C) and in the second dimension (according to the size) by 5% to 23% polyacrylamide concave gradient gel electrophoresis (125 V, 24 hours, 4°C). Iodinated high-molecular-weight protein mixture (7.1–17.0 nm; Pharmacia) was run as a standard on each gel. Electrophoretically separated samples were electrotransferred (30 V, 24 hours, 4°C) onto nitrocellulose membranes (Hybond ECL; Amersham). ApoA-I-containing particles were detected with immunopurified polyclonal anti-ApoA-I antibody (Biodesign) labeled with 125I. The presence of labeled 125I-ApoA-I was detected directly by autoradiography using Kodak XAR-2 film.
RNA Isolation and Analysis of Gene Expression by Real-Time PCR
Total RNA was isolated from liver tissues using the RNeasy mini RNA extraction kit (Qiagen) according to manufacturer’s instructions. Total RNA (200 ng) was reverse-transcribed using the QuantiTect Reverse Transcription kit (Qiagen). Real-time quantitative PCR was performed using the Quantitect SYBR Green PCR kit and QuantiTect Primer assays (Qiagen): Wwox, (QT00147735), ApoA-I (QT00110663), and Abca1 (QT00165690). All reactions were performed on an ABI PRISM 7300 Sequence Detection System (Applied Biosystems). Amplifications were performed in a 96-well plate with 50 μL reaction volumes and 40 amplification cycles (94°C, 15 s; 55°C, 30 s; 72°C, 34 s). All samples were run in triplicate and mRNA expression was taken as mean of 3 separate experiments. The relative abundance (fold change [FC] relative to control) of target mRNA was determined using the ΔΔCt method where the expression of each gene was normalized to Gapdh (QT01020908) loading control.
Using a Tissue Tearor (Biospec Products), liver tissues were homogenized on ice in Radio-Immunoprecipitation Assay buffer (20 mmol/L Tris-HCl (pH 8), 150 mmol/L NaCl, 0.5% sodium deoxycholate, 0.1% SDS, 1% Triton X-100, and 4 mmol/L EDTA) containing complete protease inhibitors (Roche Diagnostics). The homogenate was sonicated 3 times 10 sec each before centrifugation for 3 minutes at 5000 rpm, 4°C. The supernatant was used as total liver protein extracts, and protein concentrations were measured with Bradford reagent (Bio-Rad) according to manufacturer’s instructions. Equal amounts of protein were separated by SDS-PAGE, transferred to a nitrocellulose membrane, subsequently blocked with 5% skim milk and incubated with various primary antibodies (anti-ABCA1 [Novus Biologicals], anti-ApoA-I [Biodesign], anti-ANGPTL4 [Novus Biologicals], or anti-WWOX [rabbit anti-Wwox antibody8 or obtained from Cell Signaling]) and horseradish peroxidase–conjugated secondary antibodies (Jackson Biolabs). Chemiluminescence detection was performed using Western lighting plus ECL reagents (Pierce Thermo Scientific) as described by the manufacturer. Density of target bands was quantified using BioRad Imager software.
Microarray Data Analysis
Output from the Agilent Feature Extraction software was read into R, preprocessed, and tested for differential expression using functions from the Bioconductor23 package Limma.24 Specifically, the function read.maimages was used to read raw intensities into R. Quality control was performed by inspecting various diagnostic plots of the intensity distribution and correlation structure of control and regular probes. One array, JGT059, did not pass quality control and was discarded from the data set prior to preprocessing. The normexp method with an offset value of 16 was used for global background adjustment, followed by quantile normalization and a log2 transformation (functions backgroundCorrect and normalizeBetweenArrays). Within-array duplicate spots were summarized by averaging using the function avereps. The annotation for probes was retrieved from the Gene Expression Omnibus (http://www.ncbi.nlm.nih.gov/geo/), platform id GPL10787.
Using the appropriate Limma functions, a linear model was fit to each gene separately.
This linear model included the mice sex and the Wwoxhep−/− status as independent categorical variables. Moderated t tests25 on various coefficients of interest were used to test the differences between Wwoxhep−/− and wild-type (WT) mice, or male and female mice. The differential response and the male/female average response to the Wwoxhep−/− were also tested. False discovery rate (FDR) estimation was performed using the Benjamini–Hochberg method.
Ingenuity Pathway Analysis (IPA, Ingenuity Systems, http://www.ingenuity.com) was used for pathway, network, and functional analysis. For this purpose, gene-level statistical significance for differential expression was reached at P<0.05 and |FC|>1.5. The over-representation of pathways and biological functions in the lists of significantly differentially expressed genes was tested using Fisher exact test with the Benjamini–Hochberg method used for FDR estimation.
Weighted Gene Coexpression Network Analysis
The conventional differential expression data analysis was used to identify genes that are differentially expressed between different groups of mice. We also used the weighted gene coexpression network analysis (WGCNA) to identify tightly connected subsets of genes in biologically meaningful modules.26,27 WGCNA is known to alleviate the multiple testing problems associated with conventional microarray data analysis because it focuses on the relationship between modules and the clinical traits28 instead of relating thousands of genes to clinical traits.
The weighted gene coexpression network was constructed using the blockwise Modules function.26,27 The dynamic tree-cutting algorithm was used to identify modules of coexpressed genes that were correlated to 5 binary traits: knockout status (KO versus WT), sex (female versus male), sex of the knockout mice (KO females versus KO males), knockout status of male mice (male KO versus male WT), and knockout status of female mice (female KO versus female WT). Genes not classified to any given module were assigned to the gray module. The maximum block size was set at 5000 genes; a power (β) was set to 17 as this was found to satisfy the scale-free topology criteria26; the minimum module size was set at 50; and the maximum height at which the tree could be cut was set to 0.995. All the other parameters were left at default settings.29 Subsequent analyses focused on modules that included Wwox probes. The blue module included 3 Wwox probes (A_51_P110371 corresponding the Wwox isoform ENSMUST00000004756; A_55_P2015892 corresponding the Wwox isoform ENSMUST00000160862; and A_55_P2015882 corresponding the Wwox isoform ENSMUST00000109108, respectively). The turquoise module included one Wwox probe (A_55_P2152387 corresponding the Wwox isoform ENSMUST00000004756).
In the WGCNA, to systematically search for enrichments in biological gene ontology functions, the Database for Annotation, Visualization and Integrated Discovery (DAVID) was used.30 DAVID can sort a large number of genes in a given module into relevant biological annotation terms (eg, gene ontology terms). The ≈2200 gene expression probes (FC>1.5) were used as background in the DAVID analyses. DAVID further determined the enrichment scores and nominal P values (Fisher exact test) of the underlying biological term.30
Human Study Samples
The human study samples consisted of 2 large French Canadian families collected at the Cardiovascular Genetics Laboratory of the McGill University Health Center, Royal Victoria Hospital, Montreal, Canada, as described previously.31 The affection status in all families was determined using the 5th age- and sex-specific population percentiles of HDL-C.31 Lipids and lipoproteins were measured using standardized techniques as described previously.32 The research protocol was approved by the Research Ethics Board of the McGill University Health Center, and all subjects gave informed consent.
Library Construction and Resequencing
We selected 2 closely related family members per family with HDL-C levels≤5th age–sex percentile and 4 unrelated controls with HDL-C levels ≥30th percentile for targeted sequencing. The selected families also demonstrated both shared haplotypes and linkage to low HDL-C on chromosome 16q23-q24.3,33 We performed targeted resequencing±100 kb of WWOX as well as 2 other regional genes previously known to influence HDL-C levels (LCAT and CETP±100 kb) to search for other possible regional causes of low HDL-C. We used Agilent SureSelect Target Enrichment System and masked the repeat regions to avoid read alignment errors.
Resequencing Data Analysis
Each sample was sequenced on one lane of Illumina GA2 Analyzer. We used Burroughs–Wheeler Aligner34 to align sequence reads to the reference sequence (hg19) and SAMtools to make single nucleotide polymorphism (SNP) calls. Duplicates were removed, and a pileup file was generated using SAMtools.35 After quality control, the mean coverage was 110×. In the sequenced HDL-C cases and controls, we found 1965 SNPs, 1.02% of which were not present in the HapMap,36 the 1000 Genomes Project,37 and the dbSNP135 databases. Next, we filtered those variants that were shared only by the affected subjects of the 2 low HDL-C families and not present in the normal controls resequenced. Annovar was used for functional annotation.38 In these families, we did not identify any exonic or intronic CETP or LCAT variants that would have been shared by all of the sequenced low HDL-C family members and absent from the sequenced individuals with normal HDL-C levels. Association analysis in the extended low HDL-C families was performed using a measured genotype approach using the Polygenic-QTL option of Mendel,39 using continuous HDL-C levels with age and sex as covariates.
Statistical analyses were performed with GraphPad statistical software 5.0 (GraphPad Softwares Inc). Data were expressed as mean±SEM, as indicated in figure legends. Two-tailed Student’s t-test was used for comparisons between 2 groups as applicable, where indicated, and a level of significance of P<0.05 was considered statistically significant. For microarray analyses, the LIMMA R package25 was used to fit a linear model to each gene and to perform moderated t-tests on the differences between WT and Wwoxhep−/− mice, or male and female mice. Enrichment analysis for the weighted gene coexpression network study was performed using the DAVID software,30 as described in its corresponding section. The over-representation of pathways and biological functions in the lists of significantly differentially expressed genes was tested using Fisher’s exact test with the Benjamini–Hochberg method used for FDR estimation, as described in the Microarray Data Analysis section.
Resequencing of WWOX in Low HDL-C Families
We first examined the genetic association between low HDL-C and the WWOX gene. We selected 4 family members (HDL-C≤5th age–sex percentile, Table 1) from 2 French Canadian families with linkage on chr16q23-24 where the WWOX gene is located40 and 4 unrelated controls (HDL-C ≥30th percentile, Table 1) for targeted resequencing of the entire 1.1 Mb WWOX region±100 kb, in search for regional variants causing low HDL-C. We did not identify any exonic or intronic CETP or LCAT variants in these families. Lipid levels and other clinical characteristics of all members from both French Canadian families are indicated in Table I in the Data Supplement. We identified 19 variants that were shared by all sequenced cases in families 1 and 2 and absent in normal controls. We selected 7 SNPs and an INDEL based on their frequency and linkage disequilibrium to avoid typing redundant SNPs (Table 2). These variants were then investigated for cosegregation with the low HDL-C affection status in the extended low HDL-C families. All 16 affected family members, except one, shared the variant allele haplotype of rs72790052, rs4462603, rs5818121, rs67003684, rs9938194, rs16948856, and rs11647906 (Figure 1A and 1B). The rs67003684 and rs9938194 variants are less likely candidates, however, because the haplotype cosegregating to the affected subjects in family 2 contains the WT allele of these 2 variants. Using the Mendel software, we observed statistically significant evidence for association between rs72790052, rs4462603, and rs5818121 and serum HDL-C levels (ranges of P=0.0005–0.0013; β=−0.3544 to −0.3333; and SE=0.1016–0.1035) in the combined analysis of these 2 families (Table 2). These 3 variants are located in intron 5 and are rare (minor allele frequency=≈0.1–9.0%), whereas the variants rs16948856 and rs11647906 located in intron 8 are relatively common (minor allele frequency=18% and 27%, respectively). Additionally, we observed a borderline sex effect in females (P=0.059) between these segregating WWOX variants and low HDL. Preliminary results further indicate that rs72790052 was also significantly associated with WWOX expression in human adipose microarray analyses (192 subjects, data not shown). Finally, among the 8 variants, there is only one nonsynonymous variant (rs289723) in the NLRC5 gene that did not cosegregate with low HDL-C in these families (Figure 1A and 1B). Taken together, we observed a strong genetic cosegregation between the WWOX gene and the low HDL-C trait in 2 multigenerational families, confirming and complementing previous genetic associations at the chr16q23-24 locus.41
Targeted Ablation of Wwox in Mouse Livers Alters Serum Levels of Triglycerides
We were next interested in investigating whether the human genetic phenotype could translate to a physiological effect of WWOX on HDL metabolism in a mouse model. Given that hepatocytes play a major role in the formation of nascent HDL particles, we first generated Wwoxhep−/− mice to further delineate the role of WWOX in lipid metabolism. These mice were healthy, with a normal body weight, and presented histologically normal livers throughout their lifespan.
Serum levels of total cholesterol, HDL-C and TG were measured in young and old Wwoxhep−/− mice. Although targeted deletion of Wwox in hepatocytes did not seem to result in a substantial change in total and HDL-C serum concentrations (Figure IA–ID in the Data Supplement), a significant increase in TG levels was observed in older female mice, as compared with WT (P=0.0025; Figure IF in the Data Supplement). These data suggest that the Wwoxhep−/− mouse model harbors a significant sex effect on circulating TG levels and warrants more elaborate HDL-related investigations.
Gene Expression Profiling of Wwoxhep−/− Mice Predicts Roles for Wwox in HDL and Lipid Metabolic Pathways
To determine the effect of hepatic Wwox ablation on global gene expression in the liver, we performed microarray analyses on RNA from ≈100 days old mice (8 males and 8 females, 4 WT and 4 Wwoxhep−/− per group). Statistical analysis of Agilent genechip expression profiling data identified 699 probes differentially expressed (P<0.05, |FC|>1.5) between Wwoxhep−/− and WT females and 424 for males (P<0.05, |FC|>1.5), corresponding to 473 and 311 annotated genes, respectively. Wwox levels were reduced by 2.85-fold in females (P=1.46×10−4) and decreased by 2.34-fold in males (P=4.97×10−4). Hepatic deletion of Wwox resulted primarily in a significantly different sex-specific gene expression pattern, with only 24 commonly regulated genes between males and females (Figure IIA in the Data Supplement). Heatmap cluster analyses of the top 100 differentially expressed genes in both sexes are shown in Figure IIB in the Data Supplement, where significant genotype differences were also observed. To control for the sex effect, a comparison between Wwoxhep−/− male versus Wwoxhep−/− female was also performed by subtracting the baseline WT from the Wwoxhep−/− female versus male difference (Figure IIIC in the Data Supplement). The Ingenuity resource was used for pathways, network, and functional analyses of significantly regulated probes (699 in females and 424 in males with P<0.05, |FC|>1.5) between WT and Wwoxhep−/− mice. We identified the lipid metabolism function as top statistically significant annotated molecular and cellular function in females (P=1.17×10−7; Benjamini–Hochberg FDR adjusted P=4.86×10−4, Figure 2A), containing 64 genes (Table IIA in the Data Supplement) associated with 31 different subcellular functions (range P=1.17×10−7 to 2.32×10−2; Benjamini–Hochberg FDR adjusted range P=4.86×10−4 to 1.61×10−1, Table IIIA in the Data Supplement). In males, the lipid metabolism function was ranked as 7th significant function (P=2.32×10−5; Benjamini–Hochberg FDR adjusted P=2.74×10−3, Figure 2C) associated with the Wwoxhep−/− differential expression profile, containing 56 genes (Table IIB in the Data Supplement) linked to 16 subcellular functions (range P=2.32×10−5 to 7.48×10−3; Benjamini–Hochberg FDR adjusted range P=2.74×10−3 to 9.24×10−2, Table IIIB in the Data Supplement). Importantly, top canonical pathways differentially regulated between WT and Wwoxhep−/− female mice included pathways involved in Farnesoid X Receptor/Retinoid X Receptor activation (P=0.0126), atherosclerosis signaling (P=0.0147), glycerolipid metabolism (P=0.019), fatty acid (FA) biosynthesis (P=0.0173), and Liver X Receptor/Retinoid X Receptor (P=0.0477) activation (Figure 2B), among others. The last 2 of these pathways were also common to male canonical pathways, which were additionally found to be enriched in acute-phase response signaling (Figure 2D).
To better represent differential gene expression between WT and Wwoxhep−/− mice, all genes found to be associated with the lipid metabolism function in females (64 genes, Table IIA in the Data Supplement) and males (56 genes, Table IIB in the Data Supplement) were displayed in heatmaps (Figure 3A and 3B). We obtained evidence that the following key lipid genes, among others, were significantly deregulated in females: ApoA-I (P=0.0028, FC=−1.80), neutral cholesterol ester hydrolase 1 (Nche1, P=0.003, FC=1.59), FA synthase (Fas, P=0.003, FC=1.98), glycerol-3-phosphate acyltransferase (Gpam, P=0.00426, FC=1.51), endothelial lipase (Lipg, P=0.0045, FC=1.94), Sterol Regulatory Element-Binding Protein chaperone (Scap, P=0.0093, FC=−1.80), lipoprotein lipase (Lpl, P=0.014, FC=−2.08), phospholipid transfer protein (Pltp, P=0.028, FC=1.93), angiopoietin-like 4 (Angptl4, P=0.032, FC=1.64), choline kinase-α (P=0.042, FC=−1.70), and insulin induced gene 2 (Insig2, P=0.049, FC=−1.75). Although Abca1 levels did not seem statistically significant at the mRNA level, a trend toward an Abca1 decrease and an Angptl4 increase was observed in males. Further classification of lipid metabolism genes by subcellular functions in both sexes confirmed a greater number of lipid-related functions in females than in males (31 versus 16 functions, Table IIIA versus IIIB in the Data Supplement). These include regulation of cholesterol, TG and FA concentration, synthesis, and transport, suggesting that the differentially expressed lipid-related genes between WT and Wwoxhep−/− females were strongly associated with different lipid metabolic pathways, including HDL, TG, and FA metabolism (Table IIIA and IIIB in the Data Supplement). These results were validated by network pathway analyses of the 64 and 56 genes involved in lipid metabolism for females and males, respectively (Figure IIIA and IIIB in the Data Supplement). As such, we observed through several altered metabolic pathways a predicted overall decrease in HDL liver metabolism in both sexes, and a predicted increase in TG-related genes in Wwoxhep−/− females, consistent with previous serum observations (Figure IF in the Data Supplement).
Weighted Gene Coexpression Network Analysis
To identify coexpression networks correlated with the hepatic KO status, we used the WGCNA method. WGCNA clustered the ≈2200 gene expression probes (FC>1.5) from the mice microarrays into 7 distinct gene coexpression modules and a gray module that represents the noncoexpressed genes (Figure IV in the Data Supplement). We observed associations with a nominal P value of 0.03 to 3×10−9 for 5 of the 7 modules. The turquoise, black, green, blue, and red coexpression module eigengenes (ie, the average module expression values for an individual mouse) were significantly associated with the hepatic knockout status (KO versus WT), sex (female versus male), sex of the knockout mice (KO females versus KO males), and knockout status of male (male KO versus WT) and female mice (female KO versus WT) (Figure IV in the Data Supplement).
We focused our subsequent analysis on the blue and turquoise modules that included the Wwox probes. We observed that the blue coexpression module showed a trend toward a negative correlation with the knockout status (cor=−0.48 [P=0.07]), which became significant with the knockout status of the female mice (KO versus WT) (cor=−0.81 [P=3×10−4]) and to a lesser extent with the knockout status of the male mice (KO versus WT, cor=−0.62 [P=0.01], Figure IV in the Data Supplement). The turquoise module also demonstrated a similar statistically significant trend in female mice. Taken together, the WGCNA analyses also suggest that sex strongly affects the genes coexpressed with Wwox.
We used the DAVID database30 to determine if any of the underlying biological terms were enriched within the blue and turquoise coexpression modules including the Wwox probes. The enrichment analysis of the blue modules showed genes significantly enriched in biological processes, acute phase and acute-phase response (Bonferroni corrected P=0.05–4.0×10−5). The genes in the turquoise coexpression module did not show evidence for functional enrichment (P>0.05). When we further investigated the significant enrichment categories of the blue coexpression module genes for overlap with the conventional differential microarray gene expression data, we noticed that the statistically significant enrichment categories (acute phase and acute-phase response) of the blue module contain the serum amyloids (Saa), Saa1, Saa2, and Saa3 genes, also implicated in Figure 3, as well as Table II and Figure IIB in the Data Supplement. HDL is the major carrier of these Saa1 and Saa3, and Saa is associated with a proinflammatory HDL particle.42 Altogether, these findings suggest significant functional enrichment related to acute phase and acute-phase response associated with HDL-C, underlying the blue coexpression module that included the Wwox probes.
Comparison With the Conventional Differential Gene Expression Data
When comparing the WGCNA data with the conventional differential gene expression results, we observed that the blue module included 6 of the significant genes (Cgn, Hsd17b6, Insig2, Me1, Ostb, and Wwox) detected in the conventional microarray analysis between female Wwoxhep−/− and WT mice (Figure 3; Table II and Figure IIB in the Data Supplement). Similarly, the blue module included 36 of the genes detected in the conventional differential gene expression analysis between male Wwoxhep−/− and WT mice, such as Saa1, Saa2, Socs3, and Stat3. Of the blue module genes that were found to be significantly enriched for acute phase and acute-phase response categories using the DAVID enrichment analysis, Apcs, Clec3b, Cp, F13a1, Hpx, Prg4, S100a9, Saa1, Saa2, Saa3, and Stat3 were negatively correlated with the knockout status of the male mice (KO versus WT) in accordance with conventional differential analyses data (Figure 3; Table II and Figure IIB in the Data Supplement). Importantly, we also observed that the turquoise coexpression module included 50 of the genes detected in the conventional differential gene expression analysis including the key lipid genes Apoa1, Apoc2, and Lipg (Figure 3; Table II and Figure IIB in the Data Supplement). Taken together, the WGCNA and functional enrichment analyses provide a novel network of genes coexpressed with the Wwox gene and enriched for functions in acute-phase response and lipid metabolism in a sex-dependent manner.
Gene Expression Validation of HDL- and TG-Regulators in Wwoxhep−/− Mice
We next sought to validate findings from microarray and WGCNA analyses by assessing protein levels of key regulators of HDL and TG metabolism, in addition to quantitative real-time PCR validation (data not shown). One of the significantly downregulated lipid-related genes in Wwoxhep−/− female mice was ApoA-I (P=0.0028, FC=−1.8), which interaction with ABCA1 is essential for nascent HDL formation. In both male and female tissues, a significant reduction in ApoA-I protein levels was found (males 55% reduction, *P=0.0068, Figure 4A and 4D; females 50% reduction, ***P=0.00073, Figure 4B and 4E). There was also a concomitant decrease in Abca1 in males (50% reduction, *P=0.035, Figure 4A and 4D), but no significant change in females (Figure 4B and 4E). We also examined Angptl4, recently established as a potent modulator of blood plasma TG and HDL levels,43,44 and found its levels to be significantly upregulated in our Wwoxhep−/− mice microarray data (Table IIA in the Data Supplement). Consistent with mRNA levels, Angptl4 protein expression was higher in Wwoxhep−/− mice compared with the WT counterpart (60% increase in males and 35% increase in females, Figure 4C and 4F). In concordance with microarray data, these protein results point toward involvement of Wwox in both HDL and TG metabolisms.
Given these findings, we next assessed by HPLC serum lipid levels in 10 Wwoxhep−/− females and 5 Wwoxhep−/− males. Lipoprotein fraction separation confirmed previous lipid measurements (Figure I in the Data Supplement) where sole deletion of Wwox in hepatocytes did not result in significant differences in HDL-C plasma concentrations in mice of both sexes (Figure 4G and 4H). Very-low-density lipoprotein (VLDL) - associated TG levels were however significantly increased in Wwoxhep−/− females (Figure 4I and 4J), consistent with direct serum lipid measurements (Figure IF in the Data Supplement) and most importantly, with an elevated Angptl4 expression (Figure 4C and 4F). These data thus suggest that while hepatic Wwox deletion reduces HDL liver production, it may not be sufficient to lower overall circulating HDL levels (Figure 4G and 4H; Figure I in the Data Supplement), but instead, the Wwoxhep−/− mouse model harbors a strong sex-specific effect on circulating TG levels.
Impaired HDL Biogenesis in Wwox Null Mice
Having observed that Wwoxhep−/− mice show an incomplete penetrance of the HDL phenotype observed in human families with WWOX variants, we next extended our analyses to Wwox null mice. We first examined lipid levels of Wwox−/− mice, previously described as having impaired expression of key steroidogenic enzymes.10,11,20 HPLC serum characterization revealed a marked reduction in HDL-C (Figure 5A), concomitant with a significant decrease in circulating ApoA-I levels (***P=0.0002, Figure 5B and 5C). Additionally, 2D-PAGGE analysis of HDL subspecies in Wwox null mice showed significantly reduced amounts of larger α-ApoA-I-containing particles (LpAI) as well as preβ migrating subpopulations (Figure 5D). Separation of serum lipoproteins by 2D-PAGGE in heterozygous Wwox+/− mice revealed no difference in lipid profiles compared with WT mice (data not shown), as expected.11,13
We further assessed the effect of Wwox deletion in hepatic tissues from 2 day old Wwox−/− mice. Expression of key regulators of HDL biogenesis, ApoA-I and ABCA1, were examined. Levels of both Abca1 and ApoA-I mRNA were decreased in Wwox−/− compared with WT, as assessed by real-time PCR (Abca1 ***P<0.00058, Figure 5E). These results, obtained from a 129/SvjxC57BL/6J mixed background mouse model, were subsequently confirmed in hepatocytes from a FVB pure background model: mRNA levels of both Abca1 and ApoA-I were significantly decreased by 45% and 35%, respectively (Abca1 **P=0.0013, ApoA-I *P=0.0145, Figure 5F). Hepatic Abca1 and ApoA-I protein levels were also significantly reduced by 40% and 80%, respectively (Abca1 **P=0.015, ApoA-I ***P=0.0007, Figure 5G–5J). This suggests that Wwox ablation may alter endogenous production of ApoA-I and subsequent lipidation through the Abca1 transporter.
Together, these data are consistent with the observed decrease in HDL-C serum levels and nascent HDL subspecies as well as human genetic findings. Not only do Wwox−/− mice exhibit a reduction in hepatic HDL production, but they also show a systemic impairment in HDL metabolism. The HDL phenotype observed in these Wwox null mice is thus consistent with the low HDL trait in families sharing the WWOX variants allele haplotype.
In this study, we show that WWOX is involved in HDL, TG, and overall lipoprotein metabolism, using a combination of next-generation resequencing in HDL-deficient families, in vivo functional studies using liver-specific Wwoxhep−/− and total Wwox−/− mouse models, and conventional and modern-WGCNA gene microarray analyses.
First, we report a strong cosegregation of WWOX with the low HDL trait in French Canadian families with HDL-C≤5th age–sex percentile (Figure 1 and Table 2). We have previously identified a variant in the WWOX gene region to be associated with low serum HDL-C levels in a study sample comprising 9798 subjects.3 The same region has also been implicated in multiple linkage studies for HDL-C.31,45–47 Here, we determined that the minor allele haplotype ATT (rs72790052, rs4462603, and rs5818121) perfectly cosegregated with low HDL-C in families (P=0.0013–0.0005; β=−0.3544 to −0.3333), suggesting intron 5 of WWOX as the location of the functional variant. Additionally, there was a borderline significant sex effect (P=0.059) between segregating WWOX variants and the low HDL-C trait (given a limited number of individuals in our families). These findings validate the role of WWOX in determining a low-HDL phenotype in humans and propose an effect in sex-specific interactions.
Using two Wwox-deficient mouse models, we determined that in the absence of Wwox, mRNA and protein levels of key regulators of HDL metabolism are altered. Both Wwox null and Wwoxhep−/− mice demonstrated reductions in ApoA-I and Abca1 levels, critical components in reverse cholesterol transport and generation of nascent HDL particles48 (Figures 4 and 5). Interestingly, the small decrease in Abca1 mRNA levels in Wwoxhep−/− males (FC=−1.14) observed by microarray analyses translated to a significant reduction in Abca1 protein levels (50% reduction, *P=0.035, Figure 4A and 4D). Conversely, Wwoxhep−/− females displayed unchanged mRNA and protein Abca1 levels (FC=−1.04, Figure 4B and 4E). This might be due to sex-specific translational regulation. ApoA-I protein levels were, however, observed to be reduced in both Wwoxhep−/− sexes (males 55% reduction, **P=0.0068, Figure 4A and 4D and females 50% reduction, ***P=0.00073, Figure 4B and 4E), in line with decreases in total KO mice findings (Figure 5). These results in both Wwox null and Wwoxhep−/− models, coupled with serum analyses, not only suggest a key role for Wwox in HDL biogenesis but also, most importantly, suggest that ApoA-I and Abca1 might be among Wwox targets that regulate HDL-C serum concentrations. To date, limited information has been found on Wwox interacting partners in lipid metabolism although it has been shown to alter the activity of transcription factors through binding to PPxY-rich motifs.49,50
Despite consistent results in liver tissues of both mice models, differences in serum lipid levels between total and liver-specific Wwox KO animals were observed. Wwox null mice have significantly lower HDL-C and serum ApoA-I levels (Figure 5A–5C), confirming previous observations10,11,20 and correlating with decreased α1-LpAI and preβ migrating particles (Figure 5D), consistent with human disorders of HDL biogenesis.51 By contrast, liver-specific Wwox deletion did not result in a substantial change in serum cholesterol or HDL levels (Figure 4G and 4H; Figure IA–ID in the Data Supplement). It was determined that circulating levels of HDL-C were subject to small variations between individual Wwoxhep−/− mice (Figure I in the Data Supplement). Instead, a significant increase in overall TG levels and VLDL-TG content in serum lipoproteins was observed in older Wwoxhep−/− female mice, by both plasma lipid measurements and HPLC lipoprotein profiling (Figure 4I and 4J; Figure IF in the Data Supplement). These results are in agreement with previous human genetic studies documenting an association between Wwox and TG levels.6
Specific disruption of Wwox in the liver revealed important roles in lipid metabolism. In addition to identifying the lipid metabolism function as the most significantly deregulated function in female Wwoxhep−/− mice, and also prominently affected in males, microarray analyses identified several lipid-related canonical pathways differentially expressed between WT and Wwoxhep−/− female and male mice (Figure 2). Furthermore, when specifically examining the 64 female and 56 male genes (P<0.05, |FC|>1.5) associated with the lipid metabolism (Figure 3A and 3B; Table IIA and IIB in the Data Supplement), we observed multiple genes involved in cholesterol homeostasis, hydrolysis and biosynthesis of TG, and FA biosynthesis. As demonstrated through network analyses (Figure IIIA and IIIB in the Data Supplement), upregulation of these genes, such as Angptl4, Fasn, Pltp, Gpam, and Lipg, and downregulation of ApoA-I, Lpl, and Insig2 suggest global effects on several pathways in lipid metabolism, modulated by Wwox ablation in livers of both sexes (Table IIIA and IIIB in the Data Supplement). Importantly, these networks point toward a decrease in HDL metabolism in both males and females (Figure IIIA and IIIB in the Data Supplement). Complementing this, it was also observed through both conventional and WGCNA studies that male canonical pathways associated with acute-phase signaling and immune response genes were greatly affected by Wwox liver disruption (Figure 2; Figures IIIB and IV in the Data Supplement). The functional WGCNA analyses further demonstrate a novel gene network coexpressed with Wwox and enriched in lipid and acute-phase functions. This is in concert with previous findings where WWOX plays a central role in multiple signal transduction pathways.14
Microarray studies led us to pursue several genomic targets involved in lipoprotein metabolism. In addition to downregulated mRNA levels of ApoA-I and Abca1, which findings were subsequently validated through protein analyses (Figure 4A, 4B, 4D, and 4E), we also investigated Angptl4, an inhibitor of Lpl.43 In line with the array data, depletion of Wwox in liver tissues caused a 60% and 35% increase in Angptl4 protein expression in males and females, respectively (Figure 4C and 4F). In combination with other TG regulating genes, such as Lpl which was reduced 2.08-fold, Angptl4 gene and protein upregulation might be responsible for the observed increase in circulating TG levels and VLDL-TG content in Wwoxhep−/− female mice. Supporting these findings, Lichtenstein and Kersten44 showed that Angptl4 overexpression increases plasma TG by decreasing Lpl activity, converting Lpl from a catalytically active dimer to an inactive monomer, thus increasing VLDL-associated TG levels. Angptl4 overexpression thus impairs Lpl-dependent plasma TG and cholesteryl ester clearance and subsequent uptake of FAs and cholesterol into tissues, explaining the identified upregulation of FA biosynthesis pathways and TG-related genes in Wwoxhep−/− mice. Additionally, although ANGPTL4 primarily affects plasma levels of TG, the gene was recently identified in genome-wide association studies to also affect other related metabolic parameters, such as HDL metabolism.52
Sex was an important factor in determining biological roles for WWOX. Unlike the considerable difference in hepatic Abca1 protein identified in males (Figure 4A and 4D), the Abca1 levels in Wwoxhep−/− females were comparable to WT mice (Figure 4B and 4E). By contrast, the increased production of hepatic Angptl4 in Wwoxhep−/− females (Figure 4C and 4F; Table IIA in the Data Supplement) was concomitant with the observed elevated serum TG levels (Figure 4J; Figure IF in the Data Supplement), whereas Angptl4 in Wwoxhep−/− males had a modest influence on TG concentrations (Figure 4I; Figure IF in the Data Supplement). This sex-specific effect was also maintained by differentially expressed pathways in conventional microarray and WGCNA analyses (Figure 2 versus Figure IV in the Data Supplement). These data support the observation that Wwox seems to play a more prominent role in female HDL, TG and FA metabolism. This is in concordance with previous studies where WWOX was found to regulate steroid hormone pathways given its high expression in steroidogenic tissues, such as testis and ovaries, both in humans and mice.20 Altogether, our observations thus suggest a WWOX-dependent hormonal effect on the lipid metabolism.
Interestingly, and in line with these investigations, our findings are that Wwoxhep−/− mice maintain normal circulating HDL-C and ApoA-I levels despite a significant reduction in Abca1 and ApoA-I hepatic production. Although Timmins et al53 reported that hepatic Abca1 KO mice show a ≈80% decrease in plasma HDL and ApoA-I levels, indicative of the decisive role of hepatic Abca1 in HDL biogenesis, our Wwoxhep−/− model did not demonstrate these significant plasma changes. As such, in the male Wwoxhep−/− mice, despite reductions of 50% and 55% in hepatic Abca1 and ApoA-I proteins, respectively, HDL serum levels were subject to small variations between individual Wwoxhep−/− mice and, on average, remained unchanged (Figure 4A, 4D, and 4G; Figure IC and ID in the Data Supplement). These findings could be due to several reasons. First, Wwox levels were only reduced by 2.85-fold in female and 2.34-fold in male Wwoxhep−/− mice, which might be insufficient to decrease whole-body circulating HDL-C levels. Second, although our knockout mouse model was hepatocyte specific, livers contain other cells including stellate, sinusoidal endothelial, and Kupffer cells, which may express Wwox in the livers that were isolated. The effect of hepatic Wwox disruption may thus be diluted by other Wwox-expressing cells. Third, our study comprised a limited number of mice with individual variations in HDL-C plasma concentrations. Despite this number, we were able to show that WWOX is significantly involved in modulating HDL and lipid metabolism through a combination of techniques (next-generation resequencing in HDL-deficient families, in vivo animal studies, and gene microarray analyses), the results of which, we believe, validate one another. Finally, but most notably, these findings also strongly suggest that other HDL-producing tissues and biochemical and genetic regulators of HDL may compensate for HDL production and contribute to the circulating pool of HDL-C in the absence of hepatic Wwox. The intestine may thus compensate for plasma HDL biosynthesis in this mouse model, warranting additional investigations. Nonetheless, this supports the notion that the effects of WWOX extend beyond the liver, as evidenced by its involvement in multiple genetic pathways (Figure 2; Figure III and Table IIIA and IIIB in the Data Supplement).
Importantly, despite the plasma HDL-C levels in Wwoxhep−/− mice, our microarray findings report both through mRNA, subsequent Abca1 and ApoA-I protein levels, as well as genomic pathway analyses, an overall decrease in HDL production (Figure III in the Data Supplement). These observations led us to extend our analyses to a total Wwox−/− mouse model, where Wwox was ablated in all tissues. Although it is known that whole-body Wwox deletion leads to growth retardation, resulting in early death by 4 weeks of age, it was identified that homozygous Wwox-null pups were healthy and indistinguishable from their WT littermates ≤4 days postpartum, with no histological lesions in the liver or other organs.13 In our studies, we thus assessed the effect of Wwox deletion in hepatic tissues from 2 day old Wwox KO mice. Our results demonstrate a considerable decrease in ApoA-I and Abca1 expression levels, and subsequent HDL-C concentrations (Figure 5), underlining a critical role for Wwox in lipid metabolism. Several HDL-producing sources might therefore be affected by Wwox ablation and thus be responsible for the decrease in HDL.
Using the Wwox null model, we were also able to complement and translate the human genetic phenotype observed in the French Canadian families to a physiological effect of WWOX on HDL metabolism. As such, the markedly low levels of circulating HDL and ApoA-I in total Wwox KO mice, strongly supported by impaired key regulators of HDL biogenesis, are consistent with the human observations of a region-wide association between WWOX variants and decreased plasma HDL-C. Specifically, the WWOX variants may account for the low-HDL phenotype in humans, as they were found to perfectly segregate in multigenerational HDL-deficient families, correlating with a whole-body genetic association between low HDL-C and human WWOX variants, similar to the decreased HDL-C concentrations in whole-body Wwox disruption. These further extend the observation that hepatic Wwox is sufficient to alter TG metabolism in a sex-specific way but whole-body Wwox might be required to modulate circulating HDL levels. Therefore, this underlines the importance of selecting specific mouse models for future analyses and substantiates that, in addition to the liver, other HDL-producing sources might be responsible for circulating HDL-C levels.
Collectively, our data have established a physiological significant role for WWOX in lipid and lipoprotein metabolism in mouse models and human genetic studies. This may be, in part, mediated through the ABCA1/ApoA-I pathway, raising the possibility that WWOX may be involved in the complex network of cellular cholesterol homeostasis. Although WWOX had been previously linked to HDL-C in genetic association studies,3–6 this is the first study that combines human genetics, animal models, and biochemical methods to demonstrate WWOX involvement in HDL and lipid metabolism. This report is therefore a first line of functional evidence and comprehensive examination of WWOX in lipoprotein metabolism, implicating it as a novel, important, and influential modulator of HDL-C and TG levels, both in mice and humans. These findings thus emphasize the need to further elucidate the mechanisms of action of WWOX in the HDL metabolism, which may have important implications in preventing and treating atherosclerotic cardiovascular disease.
We thank the family members who participated in the study as well as Francois Lefebvre for his great help with the microarray analyses. The technical assistance of Anouar Hafiane, MSc, is also gratefully acknowledged.
Sources of Funding
This research was supported by the Canadian Institutes of Health Research (CIHR) grant MOP 97752, CIHR grant MOP 15042 and Heart and Stroke Foundation of Canada to Dr Genest, grants HL095056 and HL-28481 from the National Institutes of Health (to Dr Pajukanta), FP7 Marie Curie Reintegration Grant to Dr Aqeilan, and National Institutes of Health/National Cancer Institute (USA) grant R01 CA102444-7 to Dr Aldaz. Dr Iatan was supported by the CIHR’s Frederick Banting and Charles Best Canada Graduate Doctoral award and Dr Reddy by the American Heart Association grant 11POST7380028.
The Data Supplement is available at http://circgenetics.ahajournals.org/lookup/suppl/doi:10.1161/CIRCGENETICS.113.000248/-/DC1.
- Received July 3, 2013.
- Accepted April 23, 2014.
- © 2014 American Heart Association, Inc.
- Leduc MS,
- Lyons M,
- Darvishi K,
- Walsh K,
- Sheehan S,
- Amend S,
- et al
- Bednarek AK,
- Laflin KJ,
- Daniel RL,
- Liao Q,
- Hawkins KA,
- Aldaz CM
- Ried K,
- Finnis M,
- Hobson L,
- Mangelsdorf M,
- Dayan S,
- Nancarrow JK,
- et al
- Aqeilan RI,
- Trapasso F,
- Hussain S,
- Costinean S,
- Marshall D,
- Pekarsky Y,
- et al
- Aqeilan RI,
- Hassan MQ,
- de Bruin A,
- Hagan JP,
- Volinia S,
- Palumbo T,
- et al
- Aqeilan RI,
- Pekarsky Y,
- Herrero JJ,
- Palamarchuk A,
- Letofsky J,
- Druck T,
- et al
- Postic C,
- Shiota M,
- Niswender KD,
- Jetton TL,
- Chen Y,
- Moates JM,
- et al
- Krimbou L,
- Hajj Hassan H,
- Blain S,
- Rashid S,
- Denis M,
- Marcil M,
- et al
- Smyth GK,
- Michaud J,
- Scott HS
- Langfelder P,
- Zhang B,
- Horvath S
- Dastani Z,
- Quiogue L,
- Plaisier C,
- Engert JC,
- Marcil M,
- Genest J,
- et al
- Li H,
- Handsaker B,
- Wysoker A,
- Fennell T,
- Ruan J,
- Homer N,
- et al
- Wang K,
- Li M,
- Hakonarson H
- Tölle M,
- Huang T,
- Schuchardt M,
- Jankowski V,
- Prüfer N,
- Jankowski J,
- et al
- Lichtenstein L,
- Berbée JF,
- van Dijk SJ,
- van Dijk KW,
- Bensadoun A,
- Kema IP,
- et al
- Mahaney MC,
- Almasy L,
- Rainwater DL,
- VandeBerg JL,
- Cole SA,
- Hixson JE,
- et al
- Shearman AM,
- Ordovas JM,
- Cupples LA,
- Schaefer EJ,
- Harmon MD,
- Shao Y,
- et al
- Iatan I,
- Bailey D,
- Ruel I,
- Hafiane A,
- Campbell S,
- Krimbou L,
- et al
- Aqeilan RI,
- Donati V,
- Palamarchuk A,
- Trapasso F,
- Kaou M,
- Pekarsky Y,
- et al
- Marcil M,
- Bissonnette R,
- Vincent J,
- Krimbou L,
- Genest J
Cardiovascular disease is the leading cause of mortality worldwide. Low levels of high-density lipoprotein (HDL) cholesterol constitute an independent risk factor for heart disease, with genetic factors being the most influential HDL cholesterol determinants. Here, we advanced our understanding of the genetic regulation of HDL by characterizing a novel HDL candidate, the WW domain-containing oxidoreductase (WWOX) gene. WWOX was previously established as a tumor suppressor, but its role in cellular lipid homeostasis remained unknown. We investigated WWOX role in lipoprotein metabolism using a combination of in vivo functional studies, by next-generation resequencing in HDL-deficient French Canadian families, whole-body Wwox knockout and liver-specific mouse models, and gene microarray analyses. Our data indicate that Wwox has multiple effects on genes critical to HDL biogenesis and remodeling. We demonstrate that Wwox disruption significantly alters lipid metabolism, including cholesterol, triglycerides, and fatty acid regulation through several sex-dependent mechanisms, and contributes to the genetics of HDL cholesterol. Particularly, WWOX variants may account for a low-HDL phenotype in humans, as they segregate in multigenerational HDL-deficient families, and are consistent with Wwox KO mice findings where key regulators of HDL biogenesis are drastically impaired. These results thus represent the first functional evidence of WWOX in lipid and lipoprotein metabolism, implicating it as an important, novel modulator of HDL cholesterol and triglycerides levels, both in mice and humans. Additionally, this could signify a new link between cancer and atherosclerosis, emphasizing the need to further elucidate the mechanisms of WWOX in HDL metabolism, which may have important implications on the development and treatment of cardiovascular disease.