The etiological effect of a new low-frequency ESR1 variant on Mild Cognitive Impairment and Alzheimer’s Disease: a population-based study

ESR1 variant distribution among AD/MCI/CN

As shown in Fig. 2, this ESR1 variant was demonstrated to associate with AD risk by comparisons between AD cases and controls from our large sample collection and revealed a 3.30-fold risk of developing AD (Fig.2A; Table S3). Higher percentage of the ESR1 rs9340803 minor allele was shown in both AD and MCI cases as compared to that in CNs (4.53%, 4.23% vs. 1.37%; p<0.001), whereas the frequency didn’t differ significantly between AD and MCI cases (p=0.059). Further analyses stratified by sex and age group showed same trend existing as more minor allele carriage in AD and MCI cases (Table S4). Besides, we took APOE into consideration for further stratification, which turned out more APOEε4- AD cases (4.19%) and more APOEε4+ MCI cases (3.37%) with this ESR1 variant separately.

When comparing MCI cases to CNs solely, the disease risk was elevated by 2.08-fold with this ESR1 variant (Fig. 2A b; Table S3). There were more MCI cases with A/G heterogeneous genotype of this ESR1 variant than controls with that genotype when stratified as sex and age group (p<0.01; p<0.01). MCI cases carrying the ESR1 variant but without APOEε4 also displayed high disease risk relative to their CN counterparts (OR: 3.24 (1.74~4.20), p<0.001). However, to carriers with both the ESR1 variant and APOE ε4, there was no significant difference (p=0.190).

ESR1 variant distribution among CI/CN

AD and MCI patients were then combined together as CI cases who had amnesic memory problem in our study. As a result, these cases appeared to possess higher frequency of ESR1 rs9340803 heterozygous genotype (4.36% vs. 1.37%, p<0.001) comparing with controls, indicating the minor allele of rs9340803, like APOEε4, as a risk factor, which was manifested by a 3.18-fold of risk in the occurrence of memory problem in CI cases (Fig. 2B; Table S5). The independent effect of the G allele of this SNP apart from APOEε4 was somewhat greater than that of APOEε4 (OR= 3.23 (1.80~4.05) vs. 1.58 (1.32~1.90); Table S5). Logistic regression analysis with age, sex and APOE fixed confirmed it (Table S6). While, the joint action of the G allele and APOEε4 greatly increased the cognitive anomaly risk, with relative to individuals with both rs9340803 major allele homozygosity and non-APOEε4 (OR= 4.69 (1.39~5.89), p=0.006). Besides, the frequency of the minor allele of this SNP also differed between CI and CN subgroup members as categorized by age and sex separately, with more old and male cases with the minor allele (Table S7). Specifically, among CI cases without APOEε4, this ESR1 variant impacted the risk of disease incidence for not only females but also males (OR=2.74, p=0.048; OR=3.52, p=0.002; Table S8). Gene-gene and gene-environment interaction analyses were additionally done which further validate the joint effect of this ESR1 variant, APOEε4 and aging, with high-risk haplotype G-T-C occupying the potential of increasing disease risk to 2.46 (1.18~3.29)-fold for the elderly all and to 6.54 (0.88~10.52) if aged 70 and older (Supplementary Table S9).

AD-associated traits attributed to ESR1 rs9340803

MMSE of AD and MCI cases. The median MMSE aggregated score (median (Q)) of AD cases was 15(9, 21). There were no statistically significant differences of MMSE scored by AD cases with and without this ESR1 variant (p=0.874), with and without APOEε4 (p=0.178), or between male and female cases (p=0.103), respectively (Table S10). While divergence reached the statistical significance between older (14(9, 20)) and younger (18(13, 22)) elderly (p<0.001). Further APOEε4-stratified analysis still showed no divergences comparing carriers of this ESR1 variation with non-carriers (p=0.423; p=0.710).

The median MMSE aggregated score (median (Q)) of MCI cases was 15(11, 23). There were no statistically significant differences of MMSE scored by MCI cases with and without this ESR1 variant (p=0.488), with and without APOEε4 (p=0.078), respectively, but between male (17(13, 21)) and female (14(11, 17)) cases(p<0.001). While divergence reached the statistical significance between older (15(11, 18)) and younger (16(13, 19)) elderly (p=0.006). Further APOEε4-stratified analysis still showed no divergences comparing carriers of this ESR1 variation with non-carriers (p=0.710).

The median MMSE aggregated score (median (Q)) of CI cases was 15(11, 19). There were no statistically significant differences of MMSE scored by CI cases with and without this ESR1 variant (p=0.504), with and without APOEε4 (p=0.986), respectively, but between male (17(13, 21)) and female (14(11, 17)) cases(p<0.001). While divergence reached the statistical significance between older (15(11, 18)) and younger (16(13, 19)) elderly (p<0.001). Further APOEε4-stratified analysis still showed no divergences comparing carriers of this ESR1 variation with non-carriers (p=0.806).

Serum Aβ-oligomer concentrations. For the overall individuals, serum Aβ1-40, Aβ1-42 concentration and Aβ1-42/1-40 were observed to differ significantly among three subgroups (p<0.001, p<0.001, p<0.001). To be specific, Aβ1-40 concentration and Aβ1-42/1-40 were different in subjects from three subgroups but Aβ1-42 concentration didn’t differ between MCI cases and CNs (p=0.052; Table S11). When comparing CI cases to controls subsequently, same trend persisted (p<0.001, p<0.001, p<0.001) (Table 2). Thus, Aβ1-40, Aβ1-42 and Aβ1-42/1-40 were indicated to potentially constitute the serum Aβ profile along AD disease continuum in our study. Whereas, only divergence of serum Aβ1-40 concentration reached the statistically significant difference for E+/- (p=0.038) subclass with higher Aβ1-40 (47.12(31.46, 53.82) pmol/L) concentration in participants carrying this ESR1 variant, which might imply Aβ1-40 as a more stable biomarker in the blood. Besides, Aβ1-40 concentrations in participants with APOEε4 genotype showed a lack of statistically significant difference in comparison with those with non-APOEε4 (p=0.010; Table S12).

Plasma cholesterol levels. As the notion that elevated plasma cholesterol is a risk factor for AD, four representative indexes of Plasma cholesterols (TC, TG, HDL-c and LDL-c) were tested. In our study, TC (p<0.001) and HDL-c (p=0.050) levels were each significantly differed among individuals from three subgroups (Table S13). As regards to further comparison between CIs and CNs, there were statistically significant divergences as for TC (p<0.001) and TG concentrations (p=0.022), respectively. While, only lower TC levels were observed in the ESR1 variant carriers as compared to those non-carriers (p=0.045; Table S14). These results didn’t change when CI cases (p=0.04) and AD cases only (p=0.009) carrying the ESR1 variant were considered (Table 3). However, there were no statistically significant difference for TC levels in MCI cases carrying the ESR1 variant with relative to the counterparts (p=0.494). Additionally, statistically significant difference was not either showed for TC level between APOEε4 carriers and non-ε4 carriers (p=0.294).

Relation of ESR1 rs9340803 with plasma cholesterol levels, serum Aβ oligomer concentrations and MMSE. Serum Aβ1-40 and Aβ1-42 concentrations separately correlated to MMSE score in our study samples (p<0.001, p<0.001). Correlation of TC level to MMSE score also existed in CI cases (p<0.001). What’s more, partial correlation analyses turned out that TC (p<0.001), TG (p<0.001) and LDL-c (p<0.001) correlated to Aβ1-40 in concentration, respectively. Positive correlations between the TC level and Aβ oligomers were noted in individuals the ESR1 variant (p<0.001) even when stratified by APOEε4 (p<0.001).

ESR1 rs9340803 G variant potentially damages ESR1 transcription

This intronic SNP residing 45bp downstream of exon4 was predicted by the in silico prediction programs to broke the binding motif for a splice auxiliary protein hnRNP H1 and generate a hnRNP A1 binding motif which would promote exon 4 skipping, impairing the normal regulation activity of intronic splicing process of ESR1 pre-mRNA, which might down-regulate the transcription of ESR1 (Fig. S1).

ESR1 rs9340803 variation affects the expression of the gene

In accordance with previous prediction, dual-luciferase reporters assay showed statistically significant decreased luciferase expression (p<0.001; Fig 3A). This result indicated decreased expression of ESR1 transfected with homozygous G allele, conferring the potential functional relevance of this ESR1 A>G variant.

In silico pathway analysis of Estrogen-ERα-cholesterol in brain

Estrogen deficiency and altered lipid profile are considered significant risk factors for AD. Cholesterol is the substrate for estrogen synthesis and the possible interactions between cholesterol and estrogens in the etiology of AD, may be influenced by the cholesterol metabolism [12]. Bioinformatic pathway analysis regarding the estrogen-ERα-cholesterol cycle was thus done on the basis of ESR1 gene function, previous study results and our preliminary results (Fig. 3). Estradiol (E2 as the most form of estrogen) originating from cholesterol by source, was recognized to bind with estrogen receptor (ERα mostly in hippocampus) at first and promote complex biological outcomes in brain regions at last, playing a protective role in neurodegenerative diseases. Cholesterol content in the cytoplasm and on the membrane was modulated in some way by key enzymes, which can be regulated by interaction of factors like estrogen response elements (EREs) with ERα directly or by estrogen after bind with activated Erα (Fig. 3B).

Targeted sequencing of 12 candidate NR genes and APOE

Twelve cholesterol metabolism-related NR genes (VDR, THRA, ESR1, ESR2, LXRB, PPARA, PPARB, PPARG, AR, GR, RXRA and RXRB) were selected for next-generation sequencing on 73 LOAD patients (Supplementary Methods). We incorporated APOE into the gene panel as well.

Candidate variants selection

To discover new, rare or low-frequency variants that are associated with LOAD, we applied several rigorous analysis steps and selected candidate SNVs enriched in LOAD samples for subsequent association tests basing on given criteria. Detailed selection process referred to Supplementary Methods.

Genotyping of 9 variants using iPLEX Gold chemistry

Candidate rare or low-frequency SNVs were genotyped on 200 LOAD cases and 200 controls using the MassARRAY Compact system (Sequenom, San Diego, CA). Quality control of genotyping was carried out afterwards.

Large scale population screening on ESR1 rs9340803 and APOE

Among multiple population from nine provinces across northern and southern China mainland, additional 2694 individuals composed of 581 LOAD cases, 1059 MCI cases and 1054 controls were screened on APOEε4 and ESR1 rs9340803 genotypes.

Detection of Aβ-oligomer concentrations in the serum

On recognition that as an important biomarker of AD, Aβ can unwittingly accumulate in the brain for years, disrupting nerve connections essential for thinking and memory, and can enter systematic blood via the blood-brain barrier, we detected the concentration of serum Aβ-oligomers exploiting the Human/Rat Amyloid (40/42) ELISA Kit (WAKO; Osaka, Japan).

Detection of blood lipid fractions levels

Given that several studies have demonstrated high concentrations of plasma cholesterol increased the risk of AD, fasting lipid fractions (total cholesterol [TC], triglycerides [TGs], high-density lipoprotein cholesterol [HDL-C] and low-density lipoprotein cholesterol [LDL-C]) were studied.

In silico damaging prediction of ESR1 rs9340803

Splicing factor analysis and damaging prediction was conducted by exploiting several online websites, that is MutationTaster (http://www.mutationtaster.org/), Sfmap (http://sfmap.technion.ac.il/) and Human Splicing Finder (http://www.umd.be/HSF3/index.html), to explore on the potential effect of ESR1 rs9340803 A to G variation.

In vitro expression assay of mutated ESR1

Firefly luciferase and renilla luciferase reporter gene expression assay and kymographs were performed to analyze the expression of ESR1 transfected with wild-type or variant RTN3 constructs on 293T cell line for preliminary functional exploration on ESR1 variation.

Bioinformatic pathway analysis

KEGG (http://www.genome.jp/kegg/pathway.html) and Gene Ontology (http://www.geneontology.org/) were utilized for in silico pathway analysis linking ESR1 to AD pathogenesis.