The N342S MYLIP polymorphism is associated with high total cholesterol and increased LDL receptor degradation in humans

Abstract

Atherosclerotic cardiovascular disease (ASCVD) affects more than 1 in 3 American adults. Hypercholesterolemia is a major treatable risk factor for ASCVD, yet many individuals fail to reach target levels of LDL-cholesterol (LDL-C) through the use of statins and lifestyle changes. The E3 ubiquitin ligase myosin regulatory light chain-interacting protein (MYLIP; also known as IDOL) is a recently identified regulator of the LDL receptor (LDLR) pathway. Genome-wide association studies (GWASs) in populations of mixed European descent have identified noncoding variants in the MYLIP region as being associated with LDL-C levels, but no underlying functional variants were pinpointed. In order to fine-map actual susceptibility variants, we studied a population demographically distinct from the discovery population to ensure a different pattern of linkage disequilibrium. Our analysis revealed that in a Mexican population, the nonsynonymous SNP rs9370867, which encodes the N342S amino acid substitution, is an underlying functional variant that was associated with high total cholesterol and accounted for one of the previous significant GWAS signals. Functional characterization showed that the Asn-encoding allele was associated with more potent LDLR degradation and decreased LDL uptake. Mutagenesis of residue 342 failed to affect intrinsic MYLIP E3 ligase activity, but it was critical for LDLR targeting. Our findings suggest that modulation of MYLIP activity can affect LDL-C levels and that pharmacologic inhibition of MYLIP activity might be a useful strategy in the treatment of dyslipidemia and ASCVD.

Figure 1. Sequencing results of the MYLIP gene.

Mexican subjects with high and low LDL-C (see Supplemental Table 1 for clinical characteristics) were sequenced for the coding region and exon-intron boundaries of the MYLIP gene. Each line represents the genotype of an individual; each column represents an identified sequencing variant. Individuals are ranked according to their LDL-C levels; low-LDL individuals fall below the red line, and high-LDL individuals above. Gray, heterozygote genotypes; black, homozygote rare genotypes; white, homozygote common genotypes. See Supplemental Table 2 for major and minor alleles and positions of the variants.

Figure 2. Association results in the MYLIP gene region (±50 kb) compared between European and Mexican ethnicities.

(A) GWAS meta-analysis results of subjects of mixed European descent (6). The colored circles represent –log₁₀ of the P values for LDL-C levels; dashed line indicates the genome-wide significance threshold (log₁₀[5 × 10^–08]). Only SNPs that were also investigated in the Mexican population are shown. (B) Association results of the Mexican dyslipidemic study sample. Colored circles represent –log₁₀ of the P values for the high TC affection status. Red circles indicate SNPs in LD (r² ≥ 0.5) with rs3757354; orange circles indicates SNPs in LD with rs9370867; green circles indicate SNPs in LD with rs2327951 in the Mexican controls. The location of the SNPs is shown on the x axis in relation to the physical location of the MYLIP gene on chromosome 6.

Figure 3. Pairwise LD of the SNPs rs3757354, rs9370867, and rs2327951 with all other SNPs in the MYLIP gene region (±50 kb) in the European and Mexican populations.

Shown is pairwise LD in r² in 120 Mexican controls (MX) and in the CEU sample. SNPs shown on the x axis are ordered according to their bp position.

Figure 4. MYLIP S342 is associated with reduced ubiquitination and degradation of the LDLR and increased LDL uptake compared with N342.

(A) Immunoblot analysis of HEK293T whole-cell lysates after cotransfection with LDLR and WT MYLIP (N342/V339), S342, I339, or C387A ring domain mutant (RING MUT) expression plasmids. (B) Immunoblot of HEK293T whole-cell lysates after cotransfection with LDLR and FLAG-tagged MYLIP N342, S342, or C387A ring domain mutant expression plasmids. (C) Immunoblot of HEK293T whole-cell lysates after cotransfection with LDLR and FLAG-tagged MYLIP N342, S342, A342, or C387A ring domain mutant expression plasmids. (D) Human MYLIP mRNA and endogenous LDLR protein expression in MYLIP^–/– MEFs stably expressing empty vector (Vect; pBabe), MYLIP N342, or MYLIP S342. Data are mean ± SEM. (E) Immunoblot of HEK293T cells cotransfected with LDLR-GFP; MYLIP N342, S342, or C387A ring domain mutant; and HA-ubiquitin expression plasmids followed by overnight immunoprecipitation of GFP with anti-GFP antibody (1 μg).