Overview

Clinical Phenotype: Alzheimer's Disease
Position: (GRCh38/hg38):Chr19:44908002 T>C
Position: (GRCh37/hg19):Chr19:45411259 T>C
Transcript: NM_000041; ENSG00000130203
dbSNP ID: rs12982192
Coding/Non-Coding: Non-Coding
DNA Change: Substitution
Expected RNA Consequence: Splicing Alteration
Reference Isoform: APOE Isoform 1
Genomic Region: Intron 3

Findings

This rare intronic variant was identified in a study of the role of APOE-I3, an APOE splice variant that retains intron 3. The variant was found in post-mortem brain tissue containing APOE-I3 levels that were 13-fold higher than average (Dieter and Estus, 2010).

The APOE-I3 transcript was first discovered in cultured neurons and mouse brain tissue, where it was proposed to act as a regulator of neuronal APOE expression (Xu et al., 2008; Mahley and Huang, 2012). In a subsequent study, APOE-I3 transcripts were found to be more abundant in the brains of Alzheimer’s disease (AD) patients, with higher levels in individuals with more severe tau and amyloid pathology, and associated with increased disease risk and progression (Chen et al., 2021). Moreover, the proportion of APOE-I3 was higher in dorsolateral prefrontal cortex tissue from individuals with AD or mild cognitive impairment compared with controls. The authors also reported an increase in APOE-I3 usage with more severe Braak pathology for neurofibrillary tangles and with higher amyloid plaque pathology as defined by CERAD staging. In addition, they found that C130R (APOE4) load correlated with APOE-I3 levels, even after adjusting for disease status and pathology. The authors speculated that expression of the APOE-I3 transcript may be regulated by APOE4 and/or involved in mediating the effect of APOE genotype. Alternatively, it may be that AD pathology drives intron 3 retention. 

Dieter and Estus discovered the c.236+50T>C variant when measuring APOE-I3 in post-mortem brain samples from AD patients and cognitively healthy controls (Dieter and Estus, 2010). They found the transcript was rare, detectable in only 12 of 56 samples of anterior cingulate tissue and, in their study, it did not correlate with AD diagnosis, gender, age, or genotype for the common APOE2, 3, and 4 isoforms. In the samples with detectable APOE-I3, levels were low, constituting, on average, about 0.22 percent of total APOE mRNA. In one sample, however, it was much higher, reaching 2.8 percent.

This outlier sample belonged to a 91-year-old who had been cognitively healthy before death despite some AD neuropathology (Braak stage II). To identify genetic variants that might underlie the increased APOE-I3 levels, Dieter and Estus sequenced the APOE genes between exons 2 and 4, including the intervening introns 2 and 3. They identified four APOE variants, three common ones, including APOE4, and a rare one, c.236+50T>C. The latter, within the retained intron of APOE-I3, was absent from the other 55 brain samples, suggesting it caused the elevated intron 3 retention. Consistent with these findings, the variant was found over-represented in APOE-I3 transcripts.

The frequency of this variant in the gnomAD database is 0.000058 (Sep 2022, gnomAD v2.1.1). Most carriers were of non-Finnish European ancestry.

Biological Effect

This variant appears to modulate intron 3 splicing, promoting its retention. Whether the variant is the sole mediator of this effect, however, remains uncertain. As noted by Dieter and Estus, c.236+50T>C failed to alter splicing in transfected cells (unpublished observations). Also, the ~13-fold increase in APOE-I3 was greater than expected based on its allelic ratio (Dieter and Estus, 2010). The authors suggest these discrepancies might be due to technical limitations, and/or the lack of potentially relevant biological factors in their in vitro experiments.

The effect of the elevated levels of APOE-I3 RNA in the carrier also remains unclear. The authors speculate it could have modestly reduced APOE4 expression. APOE-I3 encodes a putative amino-terminal fragment, 79 amino acids in length, which proved undetectable in transfected cells and mouse brain (Xu et al., 2008). Nearly all APOE-I3 transcripts appear to be retained in the nucleus, but somehow avoid rapid degradation by nonsense-mediated decay (Dieter and Estus, 2010). In the carrier, this might have caused a modest loss of ApoE4 protein, since most APOE-I3 RNA appeared to be derived from the APOE4-bearing chromosome. Although APOE-I3 represents only about three percent of total APOE mRNA in the carrier, it could affect 5-6 percent of the expression of the APOE4 allele.

APOE-I3 might act as a regulator of neuronal APOE expression (Xu et al., 2008; Mahley and Huang, 2012). In transfected primary neurons, ApoE expression increased when intron 3 was removed from a genomic DNA construct and decreased when intron-3 was inserted into a cDNA construct. Also, in mice injected with the excitotoxic compound kainic acid, APOE-I3 was increased in morphologically normal hippocampal neurons but reduced in degenerating hippocampal neurons, while ApoE mRNA levels changed in the opposite direction.

The regional expression of APOE-I3 in the central nervous system may also offer clues to its physiological significance. According to Chen and co-workers, the CNS tissues that express the highest levels of the transcript include the hippocampus, substantia nigra, and spinal cord, regions particularly vulnerable to neurodegeneration (Chen et al., 2021). Dieters and Estus did not find APOE-I3 expression to correlate with the neuronal marker synaptophysin, although they noted that the very low levels of APOE-I3 expression may have prevented them from detecting such a correlation (Dieter and Estus, 2010).  Interestingly, Xu and colleagues reported it was absent from cultured astrocytes, the major producers of ApoE in the brain (Xu et al., 2008).

Yet another clue to APOE-I3’s relevance to brain function is its low level of genetic variation in humans, coupled to its poor conservation in primates (Chen et al., 2021). Genetic regions with these characteristics, also known as constrained, non-conserved regions (CNCRs), are thought to have undergone pronounced human-specific selection and appear to be enriched for sequences involved in brain-specific functions. APOE has a particularly high density of these regions, with the highest density mapping to intron 3.

This variant's PHRED-scaled CADD score (4.67), which integrates diverse information in silico, did not reach 20, a commonly used threshold to predict deleteriousness (CADD v.1.6, Nov 2022).

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References

Mutations Citations

1. APOE C130R (ApoE4)

Paper Citations

1. Dieter LS, Estus S. Isoform of APOE with retained intron 3; quantitation and identification of an associated single nucleotide polymorphism. Mol Neurodegener. 2010 Sep 7;5:34. PubMed.
2. Xu Q, Walker D, Bernardo A, Brodbeck J, Balestra ME, Huang Y. Intron-3 retention/splicing controls neuronal expression of apolipoprotein E in the CNS. J Neurosci. 2008 Feb 6;28(6):1452-9. PubMed.
3. Mahley RW, Huang Y. Apolipoprotein e sets the stage: response to injury triggers neuropathology. Neuron. 2012 Dec 6;76(5):871-85. PubMed.
4. Chen Z, Zhang D, Reynolds RH, Gustavsson EK, García-Ruiz S, D'Sa K, Fairbrother-Browne A, Vandrovcova J, International Parkinson’s Disease Genomics Consortium (IPDGC), Hardy J, Houlden H, Gagliano Taliun SA, Botía J, Ryten M. Human-lineage-specific genomic elements are associated with neurodegenerative disease and APOE transcript usage. Nat Commun. 2021 Apr 6;12(1):2076. PubMed.

Further Reading

No Available Further Reading