Overview

Clinical Phenotype: Multiple Conditions
Position: (GRCh38/hg38):Chr19:44909080 G>A
Position: (GRCh37/hg19):Chr19:45412337 G>A
Position: (GRCh38/hg38):Chr19:44909083 G>A
Position: (GRCh37/hg19):Chr19:45412340 G>A
Transcript: NM_000041; ENSG00000130203
dbSNP ID: NA
Coding/Non-Coding: Coding
DNA Change: Deletion-Insertion
Expected RNA Consequence: Deletion-Insertion
Expected Protein Consequence: Deletion-Insertion
Codon Change: GAG to AAG , GAG to AAG
Reference Isoform: APOE Isoform 1
Genomic Region: Exon 4

Findings

In this variant, two contiguous glutamates in ApoE’s lipid-binding region are substituted by lysines. Although several studies have examined its association with conditions related to lipid metabolism, few have examined it in a neurological context.

One study found three heterozygous carriers amongst 344 Koreans with memory complaints: one diagnosed with Alzheimer’s disease (AD) with small vessel disease, another diagnosed with subcortical ischemic vascular dementia described as possible AD with small vessel disease, and a third suffering from subjective cognitive impairment (Youn et al., 2017).

The AD patient was a 76-year-old man with memory loss, difficulty naming objects, and visuospatial dysfunction starting at age 75. Brain MRI revealed hyperintensities in periventricular and deep white matter and diffuse brain atrophy, particularly in the hippocampus. The patient with possible AD was an 80-year-old woman experiencing memory impairment and delusions which started at age 78. In this case, brain MRI showed moderate to severe white matter hyperintensities surrounding the ventricles and centrum semiovale, as well as multiple lacunar infarctions with diffuse brain atrophy. Lastly, the patient with subjective memory loss was a 61-year-old woman whose performance on cognitive tests was normal and whose brain MRI failed to reveal any atrophy or other abnormality. She had no family history of dementia. All three carriers were APOE3 homozygotes. None of 345 healthy controls carried the E262_E263delinsKK variant, nor was it present in 622 individuals in the KRGD Korean variant database.

The variant was also reported in two stroke patients, a 35-year-old Japanese man who also suffered from hypercholesterolemia and hypertension and a 74-year-old Japanese woman who had myocardial infarction. These patients were among the first carriers to be reported in the literature, together with two other individuals with no apparent neurological disorders (Yamamura et al., 1984).

Although rare worldwide, this variant is relatively common in individuals of East Asian ancestry, particularly in Japanese populations. For example, in one Japanese cohort the variant was found at a frequency of 0.007 (Matsunaga et al., 1995) and in the Japanese subset of the HGVD variant database it is reported at a frequency of 0.01 (Matsunaga et al., 2020). In the ExAC variant database, the global frequency was 0.0004.

Non-neurological Findings

Several studies have found a link between this variant and increased plasma lipid levels and cardiovascular disease, starting with the discovery of its encoded protein in Suita, Japan (Yamamura et al., 1984). In a study of 127 patients with lipid abnormalities and/or cardiovascular disease, Yamamura and colleagues identified four unrelated individuals whose ApoE proteins presented an unusual pattern upon isoelectric focusing. The pattern included multiple bands that migrated to more basic positions compared with the common isoforms ApoE2, 3, and 4. The most positively charged band migrated to position seven and, because it was discovered in Suita, it was named apo E-Suita (E-VII). Using neuraminidase treatment and two-dimensional electrophoresis, the authors found that this protein was unsialylated, and the other unknown bands appeared to be sialylated forms of the same protein. All four patients expressing these proteins had hyperlipidemia. In addition, two had stroke and two had myocardial infarction, one of whom also had diabetes mellitus. One patient had mild diabetes and chronic hepatitis. The authors did not detect the protein in 100 healthy controls.

Although this first study did not include DNA sequencing, the authors suspected a genetic alteration based on the ApoE isoelectric profiles and clinical phenotypes of family members (Yamamura et al., 1984). Indeed, a subsequent publication pinpointed the mutation in a Japanese patient with a similar ApoE isoelectric migration pattern (Maeda et al., 1989). This individual had hypertriglyceridemia and diabetes mellitus. Two additional carriers had diabetes or impaired glucose tolerance, elevated triglycerides, and in one case, elevated cholesterol.

Since then, the variant has been reported in multiple Japanese individuals with altered blood lipid profiles (Tajima et al., 1989; Ueyama et al., 1994; Matsunaga et al., 1995; Yanagi et al., 1997; Arai et al., 2014; Matsunaga et al., 2020), heart disease (Yamamura et al., 1990 2398626; Ueyama et al., 1994; Yanagi et al., 1997), and diabetes (Ueyama et al., 1994; Matsunaga et al., 2020).

Of note, a study of 1,138 Japanese patients suffering from familial hypercholesterolemia (FH) identified the mutation in 2.5 percent (29) of these patients, a much greater prevalence than in the general population (Tada et al., 2021).  Focusing on patients without a known FH mutation (e.g., in the LDL receptor), the prevalence was even greater, 3.9 percent. Patients with the E262_E263delinsKK variant had higher median low-density lipoprotein (LDL) cholesterol and triglyceride levels compared with those without it. In addition, LDL cholesterol levels in individuals with both a pathogenic mutation in an established FH gene and the E262_E263delinsKK variant were higher than in those with only an FH mutation.

Not all carriers, however, express these phenotypes. In the largest study of E262_E263insdelKK carriers, including 18 heterozygotes, only five subjects had high concentrations of cholesterol and eight had elevated triglycerides (Matsunaga et al., 1995). Age may be an important factor. For example, Yanagi and colleagues reported that in their study of 12 heterozygous carriers, all seven of the individuals who had hyperlipidemia were over 40 years of age (Yanagi et al., 1997). In addition, they found that even individuals with ostensibly normal levels of lipids in blood, had abnormalities when examined in greater detail. Eleven of the 12 carriers had elevated remnant lipoproteins which are associated with atherosclerosis, in particular remnant intermediate-density lipoprotein (IDL) particles loaded with cholesterol. Co-morbidities may also contribute to the phenotypes associated with this variant. One study, for example, described two carriers with elevated cholesterol, lipid deposits under the skin known as xanthomas, and coronary artery disease. Each of these individuals had two children who also carried the mutation yet were unaffected. The authors speculated that the phenotypes might have been induced by the probands’ diabetic conditions (Ueyama et al., 1994).

Biological Effect

This double substitution adds four positive charges to the ApoE lipid-binding region (Yamamura et al., 1984). How this alteration affects protein function remains unclear.  Dong and colleagues noted that the additional charges could interact with negatively charged residues in the N-terminal region of ApoE as previously proposed for the AD risk variant C130R (ApoE4) (Dong et al., 2000). Consistent with this proposal, a subsequent structural examination of E262_E263insdelKK using the RaptorX 3D program predicted such an electrostatic interaction, which the authors speculated could enhance binding to amyloid-β as has been described for ApoE4 (Youn et al., 2017). However, ApoE structure, and how it differs between the common ApoE isoforms, including ApoE4, is still under investigation (see Chen et al., 2021). 

The mutant also shares with ApoE4 a preference for associating with very low-density lipoprotein (VLDL) particles in plasma (Yamamura et al., 1999; Dong et al., 2000). In the case of ApoE4, this VLDL enrichment accelerates the particles’ clearance by hepatocytes, which in turn results in LDL receptor downregulation and elevated LDL plasma levels (Mahley et al., 2016). The previously mentioned electrostatic, long-range interactions of ApoE4 were suspected to mediate this VLDL preference (Dong et al., 1994). Thus, Dong and colleagues tested if a similar conformational mechanism might explain the VLDL preference of E262_E263insdelKK. Instead, they found evidence for a direct effect of the mutant lysines on VLDL association (Dong et al., 2000). Artificial mutants in the N-terminal domain designed to eliminate the electrostatic interactions with the mutant lysines still favored VLDL association, and even a C-terminal fragment devoid of the N-terminal domain (amino acids 210-317), retained its preference for VLDL.

The receptor-binding abilities of this mutation have also been studied, yielding mixed results. Using a competitive binding assay, Yamamura and colleagues found decreased binding of the artificially lipidated protein to the surface of human fibroblasts (23 percent of that of ApoE3; Yamamura et al., 1999). However, neither Ueyama and co-workers, who also used cultured fibroblasts, nor Dong and colleagues, who used both cell culture and solid-phase assays, detected a binding defect (Ueyama et al., 1994; Dong et al., 2000). Dong noted that Yamamura and colleagues did not report the size of their lipidated particles, nor their phospholipid:protein ratios, which might have differed from controls and contributed to their reported effect. It has been noted that E263 likely helps shield the receptor-binding region in the absence of bound lipids by forming salt bridges with R162 and R165, both in the receptor binding region. Altering E263’s charge could destabilize these interactions, favoring premature binding of lipid-free ApoE to receptors (Zhou et al., 2018).

The E262_E263insdelKK variant’s affinity for heparin appears similar to that of ApoE3 when the protein is either complexed with an artificial lipid or forms part of a VLDL particle (Yamamura et al., 1984; Dong et al., 2000). However, in its unlipidated form, the mutant’s affinity for heparin is greater compared with that of ApoE3 (Yamamura et al., 1999; Dong et al., 2000).

In one study, in silico analyses using the Mutation Taster and Polyphen 2 algorithms predicted E262_E263insdelKK had damaging effects, while FATHMM and SIFT predicted that the substitutions were tolerated (Matsunaga et al., 2020). 

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References

Mutations Citations

1. APOE C130R (ApoE4)

Paper Citations

1. Youn YC, Lim YK, Han SH, Giau VV, Lee MK, Park KY, Kim S, Bagyinszky E, An SS, Kim HR. Apolipoprotein ε7 allele in memory complaints: insights through protein structure prediction. Clin Interv Aging. 2017;12:1095-1102. Epub 2017 Jul 11 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. 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.
4. Matsunaga A, Nagashima M, Yamagishi H, Saku K. Variants of Lipid-Related Genes in Adult Japanese Patients with Severe Hypertriglyceridemia. J Atheroscler Thromb. 2020 Dec 1;27(12):1264-1277. Epub 2020 Feb 29 PubMed.
5. Maeda H, Nakamura H, Kobori S, Okada M, Mori H, Niki H, Ogura T, Hiraga S. Identification of human apolipoprotein E variant gene: apolipoprotein E7 (Glu244,245----Lys244,245). J Biochem. 1989 Jan;105(1):51-4. PubMed.
6. Tajima S, Yamamura T, Menju M, Yamamoto A. Analysis of apolipoprotein E7 (apolipoprotein E-Suita) gene from a patient with hyperlipoproteinemia. J Biochem. 1989 Feb;105(2):249-53. PubMed.
7. Ueyama Y, Nozaki S, Yanagi K, Hiraoka H, Nakagawa T, Sakai N, Jiao S, Yamashita S, Matsuzawa Y. Familial hypercholesterolaemia-like syndrome with apolipoprotein E-7 associated with marked Achilles tendon xanthomas and coronary artery disease: a report of two cases. J Intern Med. 1994 Feb;235(2):169-74. PubMed.
8. Yanagi K, Yamashita S, Hiraoka H, Ishigami M, Kihara S, Hirano K, Sakai N, Nozaki S, Funahashi T, Kameda-Takemura K, Kubo M, Tokunaga K, Matsuzawa Y. Increased serum remnant lipoproteins in patients with apolipoprotein E7 (apo E Suita). Atherosclerosis. 1997 May;131(1):49-58. PubMed.
9. Arai M, Nishimura A, Mori Y, Ebara T, Okubo M. Hypertriglyceridemia and pancreatitis in a patient with apolipoprotein E7 (p.[E244K; E245K])/E4. Clin Chim Acta. 2014 Sep 25;436:188-92. Epub 2014 Jun 9 PubMed.
10. Tada H, Yamagami K, Kojima N, Shibayama J, Nishikawa T, Okada H, Nomura A, Usui S, Sakata K, Takamura M, Kawashiri MA. Prevalence and Impact of Apolipoprotein E7 on LDL Cholesterol Among Patients With Familial Hypercholesterolemia. Front Cardiovasc Med. 2021;8:625852. Epub 2021 Apr 13 PubMed.
11. Dong J, Balestra ME, Newhouse YM, Weisgraber KH. Human apolipoprotein E7:lysine mutations in the carboxy-terminal domain are directly responsible for preferential binding to very low density lipoproteins. J Lipid Res. 2000 Nov;41(11):1783-9. PubMed.
12. Chen Y, Strickland MR, Soranno A, Holtzman DM. Apolipoprotein E: Structural Insights and Links to Alzheimer Disease Pathogenesis. Neuron. 2021 Jan 20;109(2):205-221. Epub 2020 Nov 10 PubMed.
13. Yamamura T, Dong LM, Yamamoto A. Characterization of apolipoprotein E7 (Glu(244)-->Lys, Glu(245)--->Lys), a mutant apolipoprotein E associated with hyperlipidemia and atherosclerosis. J Lipid Res. 1999 Feb;40(2):253-9. PubMed.
14. Mahley RW. Apolipoprotein E: from cardiovascular disease to neurodegenerative disorders. J Mol Med (Berl). 2016 Jul;94(7):739-46. Epub 2016 Jun 9 PubMed.
15. Dong LM, Wilson C, Wardell MR, Simmons T, Mahley RW, Weisgraber KH, Agard DA. Human apolipoprotein E. Role of arginine 61 in mediating the lipoprotein preferences of the E3 and E4 isoforms. J Biol Chem. 1994 Sep 2;269(35):22358-65. PubMed.
16. Zhou Y, Mägi R, Milani L, Lauschke VM. Global genetic diversity of human apolipoproteins and effects on cardiovascular disease risk. J Lipid Res. 2018 Oct;59(10):1987-2000. Epub 2018 Aug 3 PubMed.

Further Reading

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