Overview

Clinical Phenotype: Blood Lipids/Lipoproteins, Hyperlipoproteinemia Type III
Position: (GRCh38/hg38):Chr19:44908786 A>C
Position: (GRCh37/hg19):Chr19:45412043 A>C
Transcript: NM_000041; ENSG00000130203
dbSNP ID: rs121918394
Coding/Non-Coding: Coding
DNA Change: Substitution
Expected RNA Consequence: Substitution
Expected Protein Consequence: Missense
Codon Change: AAG to CAG
Reference Isoform: APOE Isoform 1
Genomic Region: Exon 4

Findings

This mutation results in hyperlipoproteinemia type III (HLPP3), also known as familial dysbetalipoproteinemia, which is characterized by elevated cholesterol and triglyceride levels in blood, and early onset atherosclerosis and heart disease. Several studies have revealed that its inheritance is dominant with high penetrance.

The variant was first identified in an American HLPP3 patient whose ApoE proteins migrated to the positions of the common isoforms ApoE3 and R176C (ApoE2) upon isoelectric focusing (Rall et al., 1983). Treatment with cysteamine to assess the proteins’ cysteine content, however, revealed that, unlike the common ApoE2 protein, the patient’s ApoE2-migrating species had one, instead of two, cysteine residues. Using amino acid sequence analyses, the authors then determined that the ApoE2-like protein corresponded to ApoE3 with a substitution of glutamine for lysine at position 164.

In a subsequent report, the DNA sequence and inheritance pattern were described (Smit et al., 1990). The study included three Dutch families, with 24 individuals, 15 mutation carriers, and 12 HLPP3 patients. Segregation of the mutation with disease was high, with 12 of the 15 carriers having HLPP3 and none of the non-carriers expressing disease. Of the three unaffected carriers, one was only 18 years old (although HLPP3 onset occurs over a wide range of ages, it most commonly develops in early adulthood).

The mutation was later reported as being “invariably” associated with the expression of HLPP3, but with a variable phenotype (De Knijff et al., 1994). Examining 40 Dutch carriers from six families, De Knijff and colleagues found that all carriers had increased levels of ApoE in plasma, and elevated cholesterol and triglycerides in the very low-density lipoprotein (VLDL) fraction. However, these measurements varied between patients. The authors calculated that carrier status accounted for 57 percent of the total variance of the ratio of cholesterol in VLDL and in intermediate density lipoproteins (IDL) to plasma triglycerides, and 71 percent of the total variance of plasma ApoE levels.

This same group also noted that the HLPP3 phenotype of K164Q carriers was somewhat different than that of patients with other APOE variants, including classic HLPP3 caused by APOE2 homozygosity (Mulder et al., 1994, De Knijff et al., 1994, Zhao et al., 1994). Most notably, in K164Q carriers, the increase in plasma cholesterol was mainly confined to the VLDL fraction, whereas in typical HLPP3, the levels of cholesterol in the IDL fraction are also increased, with the IDL cholesterol to triglyceride ratio being substantially elevated (see Biological Effect below).

This variant is very rare, with a single heterozygote of European ancestry reported in the gnomAD variant database (gnomAD v2.1.1, May 2022).

Biological Effect

K164Q appears to disrupt several ApoE functions, including receptor binding, heparan sulfate proteoglycan (HPSG) binding, and lipolysis. Given its location in the ApoE receptor-binding region, it is not surprising that K164Q competes poorly with wildtype ApoE3 for binding to the surface of human fibroblasts. However, it performs much better than the ApoE2 isoform: the affinity of K164Q is approximately 40 percent of that of ApoE3, while ApoE2's affinity is less than 2 percent of ApoE3's (Rall et al., 1983). Since APOE2 is recessive with incomplete penetrance and K164Q is dominant with high penetrance, the authors noted that this finding highlights the lack of a simple correlation between the severity of the binding defect and HLPP3 risk.

Studies of ApoE structure suggest that K164, together with K161, interact directly with ligand-binding repeat domains found in members of the LDL receptor family (Guttman et al., 2010). Lipid binding may induce a conformational change that exposes K164 and K161 and provides a more positive electrostatic potential, both required for receptor binding (Lund-Katz et al., 2000).

Interestingly, K164Q’s effects on receptor binding may differ depending on its ApoE backbone. In vitro experiments with ApoE fragments bound to the artificial lipid DMPC suggest K164 has an increased PK(a) when it is on an ApoE2, versus ApoE3 or ApoE4, background, arising from a reduction in the positive electrostatic potential of its microenvironment (Lund-Katz et al., 2001). Interestingly, an artificial substitution at this same site, K164A, substantially reduced binding of ApoE4 to the microglial leukocyte immunoglobulin-like receptor B3 (LilrB3), a receptor that binds to ApoE4 more strongly than to ApoE3 or ApoE2 and activates pro-inflammatory pathways (Zhou et al., 2023).

K164 is also within the heparin-binding site of ApoE, lining the shallow groove that binds and makes direct contact with the sulfo groups of HSPGs (Libeu et al., 2001, Saito et al., 2003). At pH 7.4, the mutation appears to modestly reduce heparin binding, to 93 percent of that of wildtype ApoE3, but at pH 5.0, the effect is greater, knocking it down to 77 percent. Indeed, nuclear magnetic resonance studies showed that, in lipid-laden wildtype ApoE, K164 had an unusually low pK(a) value, indicating high positive electrostatic potential around this residue. The authors hypothesized the ionization state of K164 is critical for heparin binding and noted the potential importance of H158 and D172, as well as a heparin carboxyl group, in defining the ionic microenvironment. A subsequent study suggested heparin binding is a two-step process, with this mutation disrupting the first step which involves electrostatic interactions with rapid kinetics (Futamura et al., 2005). Also of note, when complexed to the artificial lipid DMPC, K164Q binds more strongly to heparin than wildtype ApoE3 (Saito et al., 2003).

This mutation has also been reported to disrupt lipolysis. Under physiological conditions, lipoprotein lipase breaks down triglycerides in VLDLs transforming them into IDLs as they release free fatty acids. Havekes and colleagues speculated that the elevation in VLDL observed in patients could be due to a reduction of this lipase activity (Mulder et al., 1994, de Knijff et al., 1994). To test the idea, the authors generated ApoE knockout mice expressing either K164Q or wildtype ApoE3. As predicted, mice expressing the mutant protein had much higher triglyceride plasma levels than those expressing ApoE3, and in vitro experiments showed that their VLDL lipolysis rate was 54 percent lower than that of controls (De Beer et al., 2000).

K164 is within a region, spanning residues 161 to 173, that is highly conserved across species (Frieden et al., 2015). Moreover, K164Q’s PHRED-scaled CADD score, which integrates diverse information in silico, was above 20, suggesting a deleterious effect (CADD v.1.6, May 2022).

Research models

To create an animal model expressing this mutation, Apoe knockout mice were injected with an adenovirus containing the human K164Q mutant gene under the control of the cytomegalovirus promoter (De Beer et al., 2000).

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References

Mutations Citations

1. APOE R176C (ApoE2)

Paper Citations

1. Rall SC Jr, Weisgraber KH, Innerarity TL, Bersot TP, Mahley RW, Blum CB. Identification of a new structural variant of human apolipoprotein E, E2(Lys146 leads to Gln), in a type III hyperlipoproteinemic subject with the E3/2 phenotype. J Clin Invest. 1983 Oct;72(4):1288-97. PubMed.
2. Smit M, de Knijff P, van der Kooij-Meijs E, Groenendijk C, van den Maagdenberg AM, Gevers Leuven JA, Stalenhoef AF, Stuyt PM, Frants RR, Havekes LM. Genetic heterogeneity in familial dysbetalipoproteinemia. The E2(lys146----gln) variant results in a dominant mode of inheritance. J Lipid Res. 1990 Jan;31(1):45-53. PubMed.
3. de Knijff P, van den Maagdenberg AM, Boomsma DI, Stalenhoef AF, Smelt AH, Kastelein JJ, Marais AD, Frants RR, Havekes LM. Variable expression of familial dysbetalipoproteinemia in apolipoprotein E*2 (Lys146-->Gln) Allele carriers. J Clin Invest. 1994 Sep;94(3):1252-62. PubMed.
4. Mulder M, van der Boom H, de Knijff P, Braam C, van den Maagdenberg A, Leuven JA, Havekes LM. Triglyceride-rich lipoproteins of subjects heterozygous for apolipoprotein E2(Lys146-->Gln) are inefficiently converted to cholesterol-rich lipoproteins. Atherosclerosis. 1994 Aug;108(2):183-92. PubMed.
5. Zhao SP, Smelt AH, Van den Maagdenberg AM, Van Tol A, Vroom TF, Gevers Leuven JA, Frants RR, Havekes LM, Van der Laarse A, Van 't Hooft FM. Plasma lipoproteins in familial dysbetalipoproteinemia associated with apolipoproteins E2(Arg158-->Cys), E3-Leiden, and E2(Lys146-->Gln), and effects of treatment with simvastatin. Arterioscler Thromb. 1994 Nov;14(11):1705-16. PubMed.
6. Guttman M, Prieto JH, Handel TM, Domaille PJ, Komives EA. Structure of the minimal interface between ApoE and LRP. J Mol Biol. 2010 Apr 30;398(2):306-19. Epub 2010 Mar 19 PubMed.
7. Lund-Katz S, Zaiou M, Wehrli S, Dhanasekaran P, Baldwin F, Weisgraber KH, Phillips MC. Effects of lipid interaction on the lysine microenvironments in apolipoprotein E. J Biol Chem. 2000 Nov 3;275(44):34459-64. PubMed.
8. Lund-Katz S, Wehrli S, Zaiou M, Newhouse Y, Weisgraber KH, Phillips MC. Effects of polymorphism on the microenvironment of the LDL receptor-binding region of human apoE. J Lipid Res. 2001 Jun;42(6):894-901. PubMed.
9. Zhou J, Wang Y, Huang G, Yang M, Zhu Y, Jin C, Jing D, Ji K, Shi Y. LilrB3 is a putative cell surface receptor of APOE4. Cell Res. 2023 Feb;33(2):116-130. Epub 2023 Jan 2 PubMed.
10. Libeu CP, Lund-Katz S, Phillips MC, Wehrli S, Hernáiz MJ, Capila I, Linhardt RJ, Raffaï RL, Newhouse YM, Zhou F, Weisgraber KH. New insights into the heparan sulfate proteoglycan-binding activity of apolipoprotein E. J Biol Chem. 2001 Oct 19;276(42):39138-44. Epub 2001 Aug 10 PubMed.
11. Saito H, Dhanasekaran P, Nguyen D, Baldwin F, Weisgraber KH, Wehrli S, Phillips MC, Lund-Katz S. Characterization of the heparin binding sites in human apolipoprotein E. J Biol Chem. 2003 Apr 25;278(17):14782-7. Epub 2003 Feb 14 PubMed.
12. Futamura M, Dhanasekaran P, Handa T, Phillips MC, Lund-Katz S, Saito H. Two-step mechanism of binding of apolipoprotein E to heparin: implications for the kinetics of apolipoprotein E-heparan sulfate proteoglycan complex formation on cell surfaces. J Biol Chem. 2005 Feb 18;280(7):5414-22. Epub 2004 Dec 6 PubMed.
13. de Beer F, van Dijk KW, Jong MC, van Vark LC, van der Zee A, Hofker MH, Fallaux FJ, Hoeben RC, Smelt AH, Havekes LM. Apolipoprotein E2 (Lys146-->Gln) causes hypertriglyceridemia due to an apolipoprotein E variant-specific inhibition of lipolysis of very low density lipoproteins-triglycerides. Arterioscler Thromb Vasc Biol. 2000 Jul;20(7):1800-6. PubMed.
14. Frieden C. ApoE: the role of conserved residues in defining function. Protein Sci. 2015 Jan;24(1):138-44. Epub 2014 Dec 9 PubMed.

Further Reading

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