Overview

Clinical Phenotype: Multiple Conditions
Position: (GRCh38/hg38):Chr19:44907777 G>A
Position: (GRCh37/hg19):Chr19:45411034 G>A
Transcript: NM_000041; ENSG00000130203
dbSNP ID: rs121918392
Coding/Non-Coding: Coding
DNA Change: Substitution
Expected RNA Consequence: Substitution
Expected Protein Consequence: Missense
Codon Change: GAG to AAG
Reference Isoform: APOE Isoform 1
Genomic Region: Exon 3

Findings

This variant has been found in multiple East Asian individuals with an abnormal blood lipid profile and/or a kidney disorder.

It was first described in three unrelated middle-aged Japanese men with hyperlipidemia (Yamamura et al., 1984a). One man had myocardial infarction, another had chest pain and liver disease and, of note, the third had multiple cerebral infarctions. ApoE proteins isolated from these individuals were found to migrate on an isoelectric focusing gel to positions corresponding to the common ApoE isoforms ApoE3 and C130R (ApoE4), as well as to a position one pH unit beyond that of ApoE4. Accordingly, the new isoform was named ApoE5.

The variant was also found in family members of these individuals and, of those who were male and over 50, most had ischemic heart disease or cerebral infarction. Also, all aged carriers were hyperlipidemic, however, some non-carriers were too, making the link between the variant and the phenotype unclear. Indeed, one family was suspected of having a lipid disorder, familial combined hyperlipidemia, independently of the new APOE variant. The isoform was absent from 100 healthy controls (Yamamura et al., 1984b).

The mutation was subsequently sequenced at the DNA level in one of the original probands who had cerebral infarction at age 59 (Tajima et al., 1988) and in another Japanese individual with mild hypertriglyceridemia (Maeda et al., 1989). The latter also carried the R176C (APOE2) variant in trans.

Since then, the mutation has been reported in several Japanese patients with abnormal blood lipid profiles and/or kidney dysfunction (Matsunaga et al., 1995; Miyata et al., 1999; Kobayashi et al., 2002; Kodera et al., 2017, Sasaki et al., 2018; Takasaki et al., 2018 ). A study in which two carriers were found among 1,269 Japanese subjects suggested its frequency could be relatively high in this subpopulation (Matsunaga et al., 1995). In the gnomAD variant database, only East Asian carriers are reported: nine heterozygotes, yielding a frequency of 0.00049 in this population (v2.1.1 Apr 2022).

Only one homozygous carrier, a 53-year-old Japanese woman, has been reported (Kobayashi et al., 2002). Lipid profiling of this patient revealed elevated levels of total cholesterol, cholesterol associated with low-density lipoprotein (LDL-C), and triglycerides. Moreover, although the levels of lipoproteins APOA-I and APOA-II were within normal range, apolipoproteins B, C-II, C-III, and E were elevated. Of note, the patient also carried a heterozygous mutation in the lipoprotein lipase (LPL) gene, S447Ter. Previous studies reported mixed results for the effects of the LPL mutation on plasma lipid levels. Genotyping of the woman’s son and daughter revealed they were heterozygote carriers of the APOE E21K variant and had lipid and lipoprotein profiles similar to their mother’s, but their alterations were more modest.

Also, a study of a small Chinese Hui population reported an increased frequency of this variant in patients with essential hypertension compared with controls (Yang et al., 2014).

In addition, E21K appears to increase the risk for kidney disease. A few carriers have been found to suffer from lipoprotein glomerulopathy (LPG), a disorder in which kidney glomerular capillaries dilate and accumulate lipoprotein-rich aggregates. In some cases, the specific contribution of E21K has been difficult to assess because E21K has been found in the presence of other APOE variants previously associated with LPG. For example, a 50-year-old woman with LPG and neurofibromatosis type 1 carried E21K as well as R163P (Sendai), a known risk factor for LPG (Takasaki et al., 2018).

Similarly, LPG was diagnosed in a patient carrying E21K and R165P (Chicago), another variant previously tied to LPG, on the same chromosome (Kodera et al., 2017). Interestingly, in this case, the proband’s mother also carried both mutations, but did not develop LPG. Moreover, she, but not her child, carried the C130R (APOE4) variant in trans. Also of note, the authors identified another Japanese LPG patient carrying both mutations. Although the families were not known to be related, the mothers of both probands came from the same city. An earlier report of this patient had been described as carrying the APOE2 allele and E21K, but the R165P mutation had not been identified (Miyata et al., 1999). Interestingly, this patient received a kidney transplant which appeared healthy at the time of transplantation, but developed LPG pathology within nine months.

The only case in which E21K has been tied to kidney disease in the absence of known, potentially confounding variants involved a 51-year-old Japanese man with nephrotic syndrome (Sasaki et al., 2018). The mutation was found on an APOE3 background and, except for E21K, no other variants were present in the coding sequence of the mature protein. A renal biopsy revealed scar tissue, hypertrophy of epithelial cells surrounding capillary vessels, and infiltration of lipid-laden macrophages into the glomeruli capillaries, all consistent with a disease known as focal segmental glomerulosclerosis (FSGS). Unlike the previously reported E21K carriers with kidney pathology, this patient had no signs of the lipoprotein thrombi characteristic of LPG. Because the patient’s parents were deceased, the authors were unable to determine whether the variant was inherited or arose de novo.

Biological effect

Although this mutation appears to result in hyperlipidemia, experiments testing the ability of the mutant to compete with low-density lipoprotein (LDL) for binding to cell surface receptors revealed it paradoxically has a higher affinity for the receptors than ApoE3 (Dong et al., 1990; Dong et al., 1992; Wardell et al., 1991). One possibility is that the liver downregulates LDL receptors, overcompensating for the enhanced lipoprotein uptake (Dong et al., 1990). Heparin binding, however, appears unaffected (Yamamura et al., 1984a).

Wardell and colleagues noted the mutation lies outside the receptor-binding region of ApoE, suggesting it has an indirect, structural effect on receptor affinity (Wardell et al., 1991). Indeed, an NMR study of an APOE3-like construct harboring multiple mutations to keep it from aggregating, suggested E21 participates in intra-molecular interactions, possibly hydrogen-bonding with R160 in the receptor-binding region (Chen et al., 2011). However, a subsequent study using FRET and computational simulations to study monomeric ApoE4 did not identify this bond (Stuchell-Brereton et al., 2023). 

Interestingly, the methylation status of a group of ApoE amino acids, including E21, was reported as altered in the plasma of breast cancer patients (Uen et al., 2015). The observed increase in E21 methylation was predicted to reduce intra-molecular interactions, potentially affecting receptor binding.

Also of note, E21K displays anomalous migration on SDS-polyacrylamide gel electrophoresis. It migrates as if it were 1.5-2 KDa smaller than the wild-type protein, but no indication of proteolysis or early termination was identified (Tajima et al., 1988).

This variant’s PHRED-scaled CADD score, which integrates diverse information in silico, was only 0.586, substantially below 20, a commonly used threshold to predict deleteriousness (CADD v.1.6, Apr 2022). However, some prediction tools, in particular supervised-learning algorithms, predicted it is deleterious (Pires et al., 2017 see supplementary table 2).

Nomenclature Note
There are at least four different ApoE variants, including E21K, referred to as ApoE5. They share this name because of their similar migration upon isoelectric focusing.

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References

Mutations Citations

1. APOE C130R (ApoE4)
2. APOE R176C (ApoE2)
3. APOE R163P (Sendai)
4. APOE R165P (Chicago)

Paper Citations

1. Yamamura T, Yamamoto A, Hiramori K, Nambu S. A new isoform of apolipoprotein E--apo E-5--associated with hyperlipidemia and atherosclerosis. Atherosclerosis. 1984 Feb;50(2):159-72. PubMed.
2. Yamamura T, Yamamoto A, Sumiyoshi T, Hiramori K, Nishioeda Y, Nambu S. New mutants of apolipoprotein E associated with atherosclerotic diseases but not to type III hyperlipoproteinemia. J Clin Invest. 1984 Oct;74(4):1229-37. PubMed.
3. Tajima S, Yamamura T, Yamamoto A. Analysis of apolipoprotein E5 gene from a patient with hyperlipoproteinemia. J Biochem. 1988 Jul;104(1):48-52. PubMed.
4. Maeda H, Nakamura H, Kobori S, Okada M, Niki H, Ogura T, Hiraga S. Molecular cloning of a human apolipoprotein E variant: E5 (Glu3----Lys3). J Biochem. 1989 Apr;105(4):491-3. PubMed.
5. Matsunaga A, Sasaki J, Moriyama K, Arakawa F, Takada Y, Nishi K, Hidaka K, Arakawa K. Population frequency of apolipoprotein E5 (Glu3-->Lys) and E7 (Glu244-->Lys, Glu245-->Lys) variants in western Japan. Clin Genet. 1995 Aug;48(2):93-9. PubMed.
6. Miyata T, Sugiyama S, Nangaku M, Suzuki D, Uragami K, Inagi R, Sakai H, Kurokawa K. Apolipoprotein E2/E5 variants in lipoprotein glomerulopathy recurred in transplanted kidney. J Am Soc Nephrol. 1999 Jul;10(7):1590-5. PubMed.
7. Kobayashi J, Shirai K, Murano T, Misawa Y, Tashiro J, Yoshida T, Shinomiya M. A case of hyperlipidemia with homozygous apolipoprotein E5 (Glu3-->Lys). Biochim Biophys Acta. 2002 Jun 13;1583(1):117-21. PubMed.
8. Kodera H, Mizutani Y, Sugiyama S, Miyata T, Ehara T, Matsunaga A, Saito T. A Case of Lipoprotein Glomerulopathy with apoE Chicago and apoE (Glu3Lys) Treated with Fenofibrate. Case Rep Nephrol Dial. 2017 May-Aug;7(2):112-120. Epub 2017 Jul 27 PubMed.
9. Sasaki M, Yasuno T, Ito K, Matsunaga A, Hisano S, Abe Y, Miyake K, Masutani K, Nakashima H, Saito T. Focal segmental glomerulosclerosis with heterozygous apolipoprotein E5 (Glu3Lys). CEN Case Rep. 2018 Nov;7(2):225-228. Epub 2018 May 8 PubMed.
10. Takasaki S, Matsunaga A, Joh K, Saito T. A case of lipoprotein glomerulopathy with a rare apolipoprotein E isoform combined with neurofibromatosis type I. CEN Case Rep. 2018 May;7(1):127-131. Epub 2018 Jan 22 PubMed.
11. Yang Y, Xu JR, Liu XM, Zhou J, Yang B, Li M, Wang YJ. Polymorphisms of +2836 G>A in the apoE gene are strongly associated with the susceptibility to essential hypertension in the Chinese Hui population. Genet Mol Res. 2014 Feb 27;13(1):1212-9. PubMed.
12. Dong LM, Yamamura T, Yamamoto A. Enhanced binding activity of an apolipoprotein E mutant, APO E5, to LDL receptors on human fibroblasts. Biochem Biophys Res Commun. 1990 Apr 30;168(2):409-14. PubMed.
13. Dong LM, Yamamura T, Tajima S, Yamamoto A. Site-directed mutagenesis of an apolipoprotein E mutant, apo E5(Glu3----Lys) and its binding to low density lipoprotein receptors. Biochem Biophys Res Commun. 1992 Sep 16;187(2):1180-6. PubMed.
14. Wardell MR, Rall SC Jr, Schaefer EJ, Kane JP, Weisgraber KH. Two apolipoprotein E5 variants illustrate the importance of the position of additional positive charge on receptor-binding activity. J Lipid Res. 1991 Mar;32(3):521-8. PubMed.
15. Chen J, Li Q, Wang J. Topology of human apolipoprotein E3 uniquely regulates its diverse biological functions. Proc Natl Acad Sci U S A. 2011 Sep 6;108(36):14813-8. Epub 2011 Aug 22 PubMed.
16. Stuchell-Brereton MD, Zimmerman MI, Miller JJ, Mallimadugula UL, Incicco JJ, Roy D, Smith LG, Cubuk J, Baban B, DeKoster GT, Frieden C, Bowman GR, Soranno A. Apolipoprotein E4 has extensive conformational heterogeneity in lipid-free and lipid-bound forms. Proc Natl Acad Sci U S A. 2023 Feb 14;120(7):e2215371120. Epub 2023 Feb 7 PubMed.
17. Uen YH, Liao CC, Lin JC, Pan YH, Liu YC, Chen YC, Chen WJ, Tai CC, Lee KW, Liu YR, Lin HT, Lin CY. Analysis of differentially expressed novel post-translational modifications of plasma apolipoprotein E in Taiwanese females with breast cancer. J Proteomics. 2015 Aug 3;126:252-62. Epub 2015 Jun 12 PubMed.
18. Pires AS, Porto WF, Franco OL, Alencar SA. In silico analyses of deleterious missense SNPs of human apolipoprotein E3. Sci Rep. 2017 May 30;7(1):2509. PubMed.

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