Mature Protein Numbering: L28P

Other Names: ApoE4 Freiburg, ApoE4 Pittsburgh


Clinical Phenotype: Alzheimer's Disease, Hyperlipoproteinemia Type IIa, Hyperlipoproteinemia Type IIb
Reference Assembly: GRCh37/hg19
Position: Chr19:45411110 T>C
Transcript: NM_000041; ENSG00000130203
dbSNP ID: rs769452
Coding/Non-Coding: Coding
DNA Change: Substitution
Expected RNA Consequence: Substitution
Expected Protein Consequence: Missense
Codon Change: CTG to CCG
Reference Isoform: APOE Isoform 1
Genomic Region: Exon 3


Several studies have assessed the association of this variant—tightly linked to the major Alzheimer’s disease (AD) risk factor C130R (APOE4)—with AD, and most have failed to identify increased risk beyond that conferred by APOE4. The largest study to date adjusted for APOE4, detected no association in individuals of European and European admixed ancestry (see table below, Le Guen et al., 2022).

L46P was initially found in a search for genetic modifiers of APOE4 (Kamboh et al. 1999). The authors sequenced APOE exons 1-3 and part of exon 4 in 1,118 white Americans with LOAD and 1,123 controls from three research centers in Pennsylvania, Indiana, and New York. In the AD group, they discovered 27 L46P carriers, whereas the control group had only two. Interestingly, all 29 carriers harbored the APOE4 variant as well, suggesting L46P and APOE4 were likely in cis. The elevated risk of developing AD in carriers of this haplotype seemed to stem not only from APOE4, but from L46P as well. The authors reported an approximately fivefold increase in the risk of developing AD in individuals carrying both L46P and APOE4 compared with those carrying only the APOE4 allele. Given that the variant was present in two controls, as well as in two apparently normal individuals from two AD families, the authors suggested the mutation might be pathogenic with incomplete penetrance.  

Subsequent studies confirmed the link between L46P and APOE4, but not L46P’s association with AD (see table below). One study that included data from the Kamboh et al. report as well as data from 94 AD patients and 157 controls from Northern Italy, substantiated the variant’s association with AD (Scacchi et al., 2003). However, another small study failed to detect an association in patients from Madrid, Spain (Baron et al., 2003), as did a larger study in the United States, including nearly 3,000 AD patients and 4,000 controls (Medway et al., 2014). The latter identified 36 carriers in the AD group and 20 in the control group. Although a Fisher’s exact test revealed significant association between L46P and an increased risk for LOAD, the entirety of the association could be ascribed to the presence of APOE4. Moreover, a meta-analysis of the data from all four studies indicated L46P did not substantially modify the risk associated with APOE4 (Medway et al., 2014).

Consistent with these findings, a study of two Danish cohorts totaling more than 100,000 individuals and including 811 L46P carriers, 379 who were 59 years or older, concluded the variant did not increase risk of AD beyond that due to APOE4 (Rasmussen et al., 2020). Linkage measurements were consistent with L46P most often being inherited together with APOE4 (D’=0.963, r2=0.018). In addition, a genome-wide meta-analysis including 24,087 LOAD cases and 47,793 offspring of AD parents, as well as nearly 400,000 controls, identified L46P as being associated with AD, but not “credibly causal” (Jansen et al., 2019). 

In a follow-up study by the group that originally reported L46P’s association with AD, however, the authors maintained this variant likely boosts AD risk independently of APOE4 (Fan et al., 2022). In this study, the authors re-evaluated the association in a meta-analysis of nearly 16,000 White individuals from the U.S., focusing on a subgroup of 4,138 subjects who had the APOE3/E4 genotype, including 80 L46P carriers. Because all individuals in the subgroup carried an APOE4 allele, the researchers could study the independent effect of L46P. Although the association failed to reach statistical significance (OR=1.53; 95% CI: 0.70 – 3.36; p = 0.28; see table below), the authors considered the result, together with functional data reported by others (see Biological Effects below), as supportive of L46P having an effect on AD risk independent of APOE4. They also noted that a sample of nearly 140,000 individuals with an APOE3/E4 genotype (corresponding to a cohort of more than 500,000 subjects) would be required to detect an OR of 1.5 at α = 0.05 and 80 percent power.

The frequency of L46P varies between populations. While it is relatively common in Europeans and European Americans (0.17-0.34 percent), it is rare in African and African-American populations (0.003-0.0023 percent) (gnomAD, 2019; Medway et al., 2014). Of note, L46P occurred twice among 954 alleles in HEX, a database of variants from people age 60 or older who did not have a diagnosis of a neurodegenerative disease or disease-associated neuropathology at the time of death (Apr 2022). 

Data on L46P linkage with multiple variants, across several populations, can be found in the GWAS catalog (click on “Linkage Disequilibrium” tab in the “Available data” section).

Non-neurological Associations

L46P has also been implicated in plasma lipid disruption, but the reported effects vary between studies and its relatively high frequency in European populations suggests it is likely benign. Two large studies of individuals of European ancestry reported associations with moderate elevations of cholesterol in low-density lipoprotein (LDL) particles (Rasmussen et al., 2020Richardson et al., 2022, GWAS Catalog rs769453, Sep 2022). Also, 14 percent of carriers, compared with 11 percent of the more than 100,000 individuals in the Rasmussen study, were on lipid-lowering therapy. ApoE plasma levels were moderately decreased, with mean levels slightly below the 40th percentile. A subsequent study, including 809 white carriers, was consistent with these findings, but also revealed a paradoxical 26 percent decrease in LDL cholesterol in two L46P homozygotes (Rasmussen et al., 2023). This study also reported reduced plasma ApoE levels (8 percent in heterozygotes; 35 percent in homozygotes) and increased high-density lipoprotein (HDL) cholesterol in homozygotes (11 percent). 

The variant was first linked to altered lipid physiology in a study of patients from southwestern Germany with coronary artery disease (Orth et al., 1999). The authors found an increased frequency of L46P in patients (21 of 2,874 alleles) compared with controls (10 of 4,264 alleles). Analysis of 60 heterozygotes failed to reveal a specific hyperlipoproteinemia phenotype. However, compared with carriers of only APOE4, L46P carriers had lower levels of cholesterol, apoB, and apoA-I. The variant was found in 1.8 percent of patients in a lipid clinic in Hamburg, Germany (Evans et al., 2013). The five carriers in this study had high triglyceride levels and a decreased ratio of apolipoprotein B to total cholesterol.

In a French cohort of patients with primary dyslipidemia, L46P’s frequency was 0.0016, slightly lower than that of the general population (0.0019, gnomAD v3.1.1, Nov 2021) and the French Exome Project database (0.0017) (Abou Khalil et al., 2022). In the dyslipidemia cohort, 12 carriers had elevated low-density lipoprotein (LDL) cholesterol in blood and were diagnosed with autosomal dominant hypercholesterolemia, also known as hyperlipoproteinemia type IIa (HLPP2a), and five patients had elevated triglycerides and were diagnosed with familial combined hyperlipidemia, also known as hyperlipoproteinemia type IIb (HLPP2b). Of the 12 carriers with HLPP1, ten had a strong probability of their condition being due to variants in multiple genes (weighted polygenic risk scores in deciles > VII). Two additional French L46P carriers, a 6-year-old girl and her mother, presented with hypercholesterolemia with normal triglyceride levels (Wintjens et al., 2016).

In addition, Kamboh et al. reported L46P carriers having increased high-density lipoprotein (HDL)-cholesterol levels (Kamboh et al. 1999), while Baron et al. found no significant differences in the lipid profiles of non-APOE4, APOE4, and L46P carriers, including levels of total cholesterol, HDL-cholesterol, and triglycerides (Baron et al., 2003).

Of note, seven L46P homozygotes have been described in the literature. Four German homozygotes had some form of hyperlipoproteinemia, with three also suffering from different types of hypertriglyceridemia (Orth et al., 1999). Two Danish homozygotes had decreased levels of ApoE and LDL cholesterol, as noted above, but their levels of remnant cholesterol, triglycerides, and HDL cholesterol were similar to those of non-carriers (Rasmussen et al., 2023). Lastly, a French homozygote presented with an HLPP1 phenotype similar to that of heterozygotes (Abou Khalil et al., 2022).

L46P has also been identified in carriers of other rare APOE variants: E114K and V254E (Rasmussen et al., 2023). Both compound heterozygotes had APOE3/E4 genotypes.

Biological Effect

The biological effect of this variant has been examined in several studies. Of interest to the AD field, it has been reported to promote the cellular uptake of extracellular Aβ42 peptides in human neuroblastoma cells and mouse primary neurons (Argyri et al., 2014). In addition, lipoprotein particles containing L46P increased formation of intracellular reactive oxygen species and reduced cell viability.

L46P may also affect plasma lipid metabolism. Orth et al. suggested the mutation likely disrupts the metabolism of triglyceride-rich lipoproteins and high-density lipoproteins (HDLs), without affecting the clearance of lipoprotein remnants, particles derived from lipid transport complexes that have been hydrolyzed to release fatty acids for peripheral tissue absorption (Orth et al., 1999). The authors reported higher fasting triglycerides in L46P homozygotes, but after eating, these patients cleared the remnants of triglyceride-rich proteins as efficiently as controls.

Orth et al. also found that L46P did not seem to affect in vitro binding of lipid-loaded L46P to low-density lipoprotein receptors (LDRs), nor its accumulation in LDL and very low-density lipoprotein (VLDL) complexes, as these interactions were similar to those of ApoE4 lacking the mutation. However, HDLs contained about 40 percent less of the L46P variant than of wildtype ApoE4.

L46 is located in an α-helix and the substitution to proline is expected to induce a bend in the helix that could destabilize the protein and lead to misfolding of the N-terminal domain (Argyri et al., 2014; Ittisoponpisan et al., 2018). Indeed, Argyri et al. reported that the mutant protein is thermodynamically unstable and more prone to proteolysis in vitro. Also, when interacting with lipids in vitro, the mutant protein formed particles with an abnormal structure.

The predictions of different in silico algorithms varied. SIFT analysis classified this variant as tolerated, MutationAssessor as having a medium effect, PolyPhen2 as possibly damaging, Mutation Taster as disease-causing, Provean as neutral, and Condel as damaging (Ittisoponpisan et al., 2018, Abou Khalil et al., 2022).

A study analyzing whole-genome and whole-exome sequencing data from 138,632 individuals, identified L46P as one of six APOE variants likely to have functional consequences and clinical relevance given their high prevalence in at least one population and their classification by five algorithms (SIFT, Polyphen2, MutationAssessor, PROVEAN, and DANN) as deleterious with high confidence (Zhou et al., 2018). Moreover, 11 of 16 predictive algorithms based on sequence homology, supervised-learning, protein-sequence and structure, and consensus sequence identification, predicted the substitution was damaging (Pires et al., 2017  see supplementary table 2). However, the variant’s PHRED-scaled CADD score (8.09), which integrates diverse information in silico, did not reach 20, a commonly used threshold to predict deleteriousness (CADD v.1.6, Apr 2022).

Research models

Transgenic mice have been created that express this variant under the control of the glial fibrillary acid protein promoter (Huber et al., 2000).

Note on nomenclature

Because carriers of this mutation were found to also carry the APOE4 allele, the mutation was named APOE*4 Pittsburgh (Kamboh et al. 1999). The mutation was also named APOE4 Freiburg because the same year it was described in the Pittsburgh-based study, it was reported in patients with coronary artery disease living near Freiburg, Germany (Orth et al., 1999).


Study Type Risk Allele(s) Allele Freq.
Cases | CTRL
Association Results ANCESTRY
GWAS Meta-analysis C   71,8880 | 383,378 z-score=5.59
Jansen et al., 2019
Case-control C  

11,868 | 11,934

(stage 1)

OR=1.12; [CI=0.77-1.62]     p=0.56 (adjusted for APOE4)

European/  admixed European (ADSP) Le Guen et al., 2022
Targeted Meta-analysis C 0.0085 | 0.0028 6,390 | 9,372 OR=2.87
White (American) Fan et al., 2022
Targeted Meta-analysis C 0.019 total (only APOE3/E4 carriers) 4,138 total (only  APOE3/E4 carriers) OR=1.53
[CI: 0.70–3.36]
White (American) Fan et al., 2022
Targeted C 0.012 |  0.0051 2,996 | 3,951 OR=2.39
American, European Medway et al., 2014
Targeted C 0.012 |  0.0051 2,996 | 3,951 OR=0.91
(w/APOE2,E4 as co-variates)
American, European Medway et al., 2014
Targeted Meta-analysis C (APOE4)b   1,329 | 1,401 OR=4.04c
American, European Medway et al., 2014
Targeted C 0.0169 | 0.0076 1,898 | 102,888 HR=2.36
Rasmussen et al., 2020
Targeted C 0.0169 | 0.0076 1,898 | 102,888 HR=1.16
(adjusted for APOE2/3/4)
Rasmussen et al., 2020
Targeted Meta-analysis C   1,212 | 1,280 OR=4.25
American, European Scacchi et al., 2003
Targeted C 0.0242 | 0.0018 1,118 | 1,123 OR=13.2 White
Kamboh et al., 1999
Targeted C 0.0242 | 0.0018 1,118 | 1,123 OR=5.4
(Adjusted for APOE4)
Kamboh et al., 1999

a24,087 LOAD cases; 47,793 offspring of parents with AD.
bL46P-APOE4 haplotype.
cFor comparison, APOE4 vs. the same reference group: OR=4.31 [CI=3.96-4.70].

OR=odds ratio; HR= hazard ratio. Statistically significant associations (as assessed by the authors) are in bold.

This table is meant to convey the range of results reported in the literature. As specific analyses, including co-variates, differ among studies, this information is not intended to be used for quantitative comparisons, and readers are encouraged to refer to the original papers. Thresholds for statistical significance were defined by the authors of each study. (Significant results are in bold.) Note that data from some cohorts may have contributed to multiple studies, so each row does not necessarily represent an independent dataset. While every effort was made to be accurate, readers should confirm any values that are critical for their applications.

Last Updated: 23 Aug 2023


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Mutations Citations

  1. APOE C130R (ApoE4)
  2. APOE E114K
  3. APOE V254E

Paper Citations

  1. . Association of Rare APOE Missense Variants V236E and R251G With Risk of Alzheimer Disease. JAMA Neurol. 2022 Jul 1;79(7):652-663. PubMed.
  2. . A novel mutation in the apolipoprotein E gene (APOE*4 Pittsburgh) is associated with the risk of late-onset Alzheimer's disease. Neurosci Lett. 1999 Mar 26;263(2-3):129-32. PubMed.
  3. . Screening of two mutations at exon 3 of the apolipoprotein E gene (sites 28 and 42) in a sample of patients with sporadic late-onset Alzheimer's disease. Neurobiol Aging. 2003 Mar-Apr;24(2):339-43. PubMed.
  4. . Apolipoprotein E Pittsburgh variant is not associated with the risk of late-onset Alzheimer's disease in a Spanish population. Am J Med Genet B Neuropsychiatr Genet. 2003 Jul 1;120B(1):121-4. PubMed.
  5. . ApoE variant p.V236E is associated with markedly reduced risk of Alzheimer's disease. Mol Neurodegener. 2014 Mar 10;9:11. PubMed.
  6. . APOE and dementia - resequencing and genotyping in 105,597 individuals. Alzheimers Dement. 2020 Dec;16(12):1624-1637. Epub 2020 Aug 18 PubMed.
  7. . Genome-wide meta-analysis identifies new loci and functional pathways influencing Alzheimer's disease risk. Nat Genet. 2019 Mar;51(3):404-413. Epub 2019 Jan 7 PubMed.
  8. . Investigation of the independent role of a rare APOE variant (L28P; APOE*4Pittsburgh) in late-onset Alzheimer disease. Neurobiol Aging. 2023 Feb;122:107-111. Epub 2022 Nov 18 PubMed.
  9. . Characterising metabolomic signatures of lipid-modifying therapies through drug target mendelian randomisation. PLoS Biol. 2022 Feb;20(2):e3001547. Epub 2022 Feb 25 PubMed.
  10. . APOE and vascular disease: Sequencing and genotyping in general population cohorts. Atherosclerosis. 2023 Aug 9;:117218. PubMed.
  11. . Effects of a frequent apolipoprotein E isoform, ApoE4Freiburg (Leu28-->Pro), on lipoproteins and the prevalence of coronary artery disease in whites. Arterioscler Thromb Vasc Biol. 1999 May;19(5):1306-15. PubMed.
  12. . Resequencing the APOE gene reveals that rare mutations are not significant contributory factors in the development of type III hyperlipidemia. J Clin Lipidol. 2013 Nov-Dec;7(6):671-4. Epub 2013 May 25 PubMed.
  13. . APOE Molecular Spectrum in a French Cohort with Primary Dyslipidemia. Int J Mol Sci. 2022 May 21;23(10) PubMed.
  14. . Global molecular analysis and APOE mutations in a cohort of autosomal dominant hypercholesterolemia patients in France. J Lipid Res. 2016 Mar;57(3):482-91. Epub 2016 Jan 22 PubMed.
  15. . Molecular basis for increased risk for late-onset Alzheimer disease due to the naturally occurring L28P mutation in apolipoprotein E4. J Biol Chem. 2014 May 2;289(18):12931-45. Epub 2014 Mar 18 PubMed.
  16. . Structural Biology Helps Interpret Variants of Uncertain Significance in Genes Causing Endocrine and Metabolic Disorders. J Endocr Soc. 2018 Aug 1;2(8):842-854. Epub 2018 Jun 13 PubMed.
  17. . 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.
  18. . In silico analyses of deleterious missense SNPs of human apolipoprotein E3. Sci Rep. 2017 May 30;7(1):2509. PubMed.
  19. . Characterization of transgenic mice expressing apolipoprotein E4(C112R) and apolipoprotein E4(L28P; C112R). Neuroscience. 2000;101(1):211-8. PubMed.

Other Citations

  1. HEX

External Citations

  1. GWAS catalog
  2. GWAS Catalog rs769453

Further Reading

No Available Further Reading

Protein Diagram

Primary Papers

  1. . A novel mutation in the apolipoprotein E gene (APOE*4 Pittsburgh) is associated with the risk of late-onset Alzheimer's disease. Neurosci Lett. 1999 Mar 26;263(2-3):129-32. PubMed.
  2. . Effects of a frequent apolipoprotein E isoform, ApoE4Freiburg (Leu28-->Pro), on lipoproteins and the prevalence of coronary artery disease in whites. Arterioscler Thromb Vasc Biol. 1999 May;19(5):1306-15. PubMed.

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