Triggering Receptor Expressed on Myeloid cells 2 (TREM2) is a pattern recognition receptor present on dendritic cells, monocytes, and tissue-specific macrophages [1, 2, 3]. This 230 amino acid long transmembrane glycoprotein consists of an extracellular immunoglobulin-like V type domain, a transmembrane domain, and a cytoplasmic tail [4]. Because of the short cytoplasmic tail, TREM2 acts through the intracellular adaptor molecule DNAX-activation protein 12 (DAP12), also known as TYRO protein tyrosine kinase binding protein (TYROBP) [5]. TREM2 binds to anionic lipids, high- and low-density lipoproteins, and to several apolipoproteins such as APOA1, APOA2, APOB, APOE, and APOJ (clusterin) [6, 7, 8, 9]. The molecule is implicated in a wide array of functions including cell maturation, survival, proliferation, activation, phagocytosis, and the regulation of inflammation [10]. Anti-inflammatory properties of TREM2 after TLR stimulation have been confirmed in several in vitro and in vivo studies [11, 12, 13].

In the human brain, TREM2 is primarily expressed on microglia and is involved in the phagocytosis of apoptotic neurons, and modulation of inflammation [10, 14, 15, 16]. As revealed in microglia, the reduction of TREM2 signaling increases TNF and NO synthase-2 (NOS2) transcription, while overexpression of TREM2 decreases transcription of TNF, IL1$\beta$, and NOS2 [14].

Enhanced expression of TREM2 was found in various neurodegenerative disorders such as Parkinson’s disease [17], amyotrophic lateral sclerosis [18], stroke [19, 20], traumatic brain injury [21], and Alzheimer’s disease [22, 23, 24, 25]. In Alzheimer’s disease (AD) subjects, increased TREM2 expression has been associated with the recruitment of microglia to amyloid plaques [24, 26]. In various in vitro and in vivo models, TREM2 has been associated with A$\beta$40 and A$\beta$42 uptake by microglia suggesting its effect on A$\beta$ clearance in AD models [27, 28, 29, 30, 31]. TREM2 can also increase microglial cell number, proliferation, and survival resulting in clustering of microglia to amyloid plaques [27, 32, 33].

Several TREM2 genetic variants have been identified that increase the risk of late-onset Alzheimer’s disease (LOAD). The most-well studied variant is rs75932628 C $>$ T, a single nucleotide polymorphism (SNP) in exon 2 encoding an arginine to histidine missense substitution at amino acid 47 (R47H). Two independent studies in 2013 found for the first time that heterozygous form of the TREM2 R47H is associated with LOAD risk [34, 35]. This rare TREM2 variant can increase AD risk by 2–4 fold which is comparable to the increased risk associated with one copy of the known APOE$\epsilon$4 risk allele [36, 37]. The association of the TREM2 R47H variant with AD has been observed in European [38, 39, 40, 41, 42, 43, 44, 45, 46], and Afro-American populations [47, 48]. However, the R47H variant does not seem to be associated with LOAD risk in the Asian population [49, 50, 51, 52, 53, 54]. Other TREM2 variants suggested as risk factors for developing LOAD include D87N, R62H, T96K, Y38C, L211P, and H157Y [34, 38, 47, 55, 56, 57, 58, 59, 60]. Most variants are associated with decreased cell-surface expression of TREM2 (R47H, Y38C, R136Q) or by impaired in vitro interactions with ligands like APOE, LDL, and clusterin (R47H, R62H and D87N) [27, 61, 62]. R47H carriers in AD patients also demonstrated upregulation of IFN type I response and pro-inflammatory cytokines [63]. Moreover, the R47H variant of sTREM2 is less able to bind and disaggregate oligomeric A$\beta$, which leads to A$\beta$ protofibril formation and neurotoxicity [64].

The aim of our study was to perform an association analysis between TREM2 coding variants and risk of LOAD in the Slovak population. We have analyzed 10 non-synonymous rare variants in exon 2 of the TREM2 by means of direct sequencing. To our knowledge, no such analysis has been performed in the Slovak population until now.

TREM2 is a pattern recognition receptor expressed on myeloid cells involved in the modulation of the innate immune response [3]. In the human brain, TREM2 is primarily expressed on microglia, and is involved in cell survival, chemotaxis, phagocytosis, and regulation of inflammation [10, 14, 15, 16]. It was found that TREM2 can protect against AD by binding with A$\beta$40 and A$\beta$42 monomers [27, 28, 29, 30, 31, 33]. Moreover, the soluble form of TREM2 is involved in the inhibition of A$\beta$ aggregation thus it prevents the formation of pathological amyloid plaques [64].

Rare coding variants in TREM2 were identified as risk factors for Alzheimer’s disease in several populations. In 2013 two independent studies described for the first time the association of R47H with LOAD risk [34, 35]. Since then the R47H variant has been consistently reported to increase the risk for AD across ethnicities as stated in meta-analyses [37, 46, 54, 59, 60] and large-scale GWAS analyses [71, 72].

In this study, we examined the contribution of TREM2 rare variants on risk for LOAD in the Slovak population. We have analyzed 10 non-synonymous coding variants located in TREM2 exon 2 encoding the ectodomain. Four out of 10 TREM2 mutant variants have been identified in analyzed groups, namely R47H, D87N, R62H and T96K. No statistically significant differences in the distribution of the TREM2 R47H variant were found between AD patients and the control group. This finding is due to the low frequency of the risk allele in the study population and agrees with other reports [39, 55]. In our study, the R47H variant was identified in one AD case (0.19%), but it was absent in the control group. The OR for R47H was 3.69 what is comparable with previous studies on this variant in Caucasian populations with pooled OR of 3.93 [37].

The distribution of R47H variant in AD patients seems to differ across ethnicities. The rs75932628-T was found in 0.6 to 1.4% of AD cases in the Spanish population [38, 40], 1.6 to 2.1% in the French population [73, 74], 0.74% of AD cases in the Belgian population [55], 0.4% in the Polish population [58], 1.4 to 2.5% in UK [34, 41, 42], 1.8% in the Icelandic population [43], 2.3% in the Iranian population [75], 1.8% to 6% in North Americans [56, 76, 77, 78], 0.2% in Afro-Americans [47], 1.7% in the Colombian population [48], 0.98% in the Argentinian population [59], and in 0.05% in the Japanese population [51].

Regarding other TREM2 variants in the Slovak population, D87N and R62H were found in both AD patients and controls, while T96K was identified in one control subject. No statistically significant differences in the distribution of TREM2 mutant variants were found between AD patients and the control group, as reported in other studies [34, 47, 55].

We are aware of several limitations of the current study. First, the relatively small sample sizes may reduce the power of the study to detect associations between TREM2 gene variants and the risk of late-onset Alzheimer’s disease. A larger number of LOAD patients would be desirable for replication, especially with regard to the low prevalence of most TREM variants. We assume our present-day results as useful for sample size planning in future investigations on this topic. Secondly, the estimation of age at onset may be biased by different factors such as the significance of clinical symptoms as experienced by patients.

A significant association of R47H with the risk of AD in the Caucasian population (OR = 3.93, 95% CI: 3.15–4.90, p 0.001) was confirmed in the latest meta-analysis examining 22,175 AD cases and 33,049 controls [37]. Furthermore, meta-analysis of R62H and D87N in Caucasians identified no significant association of these TREM2 variants with AD susceptibility (R62H: OR = 1.17, 95% CI: 0.90–1.52, p = 0.231; D87N: OR = 1.62, 95% CI: 0.94–2.82, p = 0.084). However, a three-stage case-control study of 48,343 AD cases and 36,790 controls revealed significant association between the R62H variant and AD risk in Caucasians (p = 1.55 $\times$ 10${}^{-14}$, OR = 1.67) [79]. A most recent meta-analysis in Caucasians also indicated that H157Y carriers were more predisposed to AD (OR = 4.22, 95% CI: 1.93–9.21, p 0.001) as compared to controls [37]. In contrast to the Caucasian population, the R47H variant has a lower frequency in the East Asian population (0.03% vs 0.08%) and thus no association of the R47H variant with AD risk has been identified [37, 52]. Similarly, no association of H157Y or L211P with AD in the East Asian population was found [52]. Concerning other TREM2 variants, S31F, R47C, G55R, L205P, and G219C were identified as novel variants in the AD patient group [56, 75, 77]. Moreover, TREM2 rs187370608 A $>$ G was significantly associated with AD susceptibility as revealed in GWAS using combined AD-by-proxy approach and case-control study (p = 1.45 $\times$ 10${}^{-16}$, p = 1.26 $\times$ 10${}^{-25}$) [80, 81].

The diminished effect of TREM2 rare coding variants on protein function has been confirmed by biochemical analyses. The most studied R47H variant showed decreased cell-surface expression and impaired ligand-binding [9, 77]. R47H variant also affects TREM2 maturation [82]. Finally, the R47H variant of soluble TREM2 is less capable of binding and disaggregating oligomeric A$\beta$, thus promoting A$\beta$-induced neurotoxicity [64]. As a consequence of loss of TREM2 function, R47H carriers showed reduced myeloid cell responses to amyloid deposition and more frequent $\alpha$-synucleinopathy [45, 83]. Some studies found that R47H carriers had a trend toward a decrease in the age of AD onset [41, 45], however, others revealed no significant association [40, 43].

Other TREM2 rare variants associated with decreased cell-surface receptor expression include R136Q, R136W Y38C, T66M, S31F, R47C, and E151K [77]. R62H and D87N showed impaired interactions with ligands, however, the T96K variant increased TREM2 affinity to their ligands [61, 84]. Y38C variant exhibited impaired TREM2 maturation and folding leading to changes in microglia morphology, loss of synaptic proteins, and reduced hippocampal synaptic plasticity in mouse models [85].

As APOE $\epsilon$4 allele belongs to known risk factors for susceptibility to AD [36, 86, 87], we also examined the combined effect of APOE$\epsilon$4 and TREM2 variants on LOAD risk. Analyses in APOE$\epsilon$4-positive and APOE$\epsilon$4-negative groups revealed no statistically significant differences in the distribution of TREM2 variants between AD patients and the control group. However, we found a somewhat higher OR for rs143332484-T (R62H) in APOE $\epsilon$4 non-carriers (1.99) than in APOE $\epsilon$4 carriers (0.62). A recent meta-analysis in non-APOE$\epsilon$4 carriers based on whole-exome sequencing data identified 4 candidate variants with strong statistical power on AD risk. They include APOE/rs7412 coding for the APOE$\epsilon$2 allele, (OR = 0.40; p = 5.46 $\times$ 10${}^{-24}$), TOMM40/rs157581 (OR = 1.49; p = 4.04 $\times$ 10${}^{-7}$) and TREM2/rs75932628 (R47H) (OR = 4.00; p = 1.15 $\times$ 10${}^{-7}$). The fourth significant variant, NSF/rs199533 (K702K), was found on chromosome 17 (OR = 0.78; p = 2.88 $\times$ 10${}^{-7}$). These candidate gene variants showed either protective or negative effects on AD risk [88].

In our study we also calculated the synergy factor value that has predicted an antagonism between TREM2 rs143332484-T (R62H) and APOE $\epsilon$4 alleles in LOAD risk. It is not clear whether the observed antagonistic interaction between TREM2 rs143332484-T (R62H) and APOE $\epsilon$4 alleles correlates with the biological events in AD subjects. APOE $\epsilon$4 has been associated with impaired binding and clearance of A$\beta$ leading to deposition of amyloid plaques in the brain [89, 90]. Moreover, APOE $\epsilon$4 has been involved in impaired synaptic integrity and plasticity [91]. Carriers of at least one APOE$\epsilon$4 allele exhibited an earlier disease onset, faster disease progression, and increased brain atrophy [36, 92, 93].

The APOE- TREM2 relationship has been studied by several authors [6, 7, 8, 94]. It was found that TREM2 is binding to APOE to enhance the phagocytosis of apoptotic neurons [6]. Regarding the TREM2 R62H variant its decreased ligand affinity has been observed [9]. It can be hypothesized that impaired ligand affinity of R62H affects phagocytosis of APOE-bound apoptotic cells by microglia contributing to AD pathology.

In our case-control study, we assessed the contribution of TREM2 rare variants on risk for LOAD in the Slovak population. We have analyzed 10 non-synonymous coding variants located in TREM2 exon 2 encoding for the extracellular domain. Four out of 10 TREM2 mutant variants have been identified in both analyzed groups, namely R47H, D87N, R62H, and T96K. R47H substitution was found only in the AD group, while T96K was present only in the controls. The OR of 3.69 in TREM2 R47H carriers suggests an increased risk of this variant for LOAD also in the Slovak population.

AD, Alzheimer’s disease; APO, apolipoprotein; A$\beta$, amyloid $\beta$; DAP12, DNAX-activation protein 12; EDTA, ethylenediamine tetraacetic acid; GWAS, genome wide association studies; LDL, low density lipoproteins; LOAD, late-onset Alzheimer’s disease; LPS, lipopolysaccharide; MoCA, Montreal Cognitive Assessment; NA, not available; OR, odds ratio; SNP, single nucleotide polymorphism; sTREM2, soluble TREM2; TLR, toll-like receptor; TNF, tumor necrosis factor; TREM2, triggering receptor expressed on myeloid cells 2; TYROBP, TYRO protein tyrosine kinase binding protein.

VD and IS—study design and manuscript writing. ZP, BV and IK—sample collection. GM and RP— analysis by direct sequencing. JJ—data analysis and interpretation. VD and AO—PCR assays. SS—data collection.

The study was approved by the Independent Ethical Committee of the Bratislava Municipality under the No. 05440/2021/HF. Informed written consent was obtained from all participants.

The authors thank all individuals for participating in this study. We gratefully acknowledge B. Misovic Faragova and Z. Nurnberger for their technical assistance.

This research was financially supported by the Scientific Grant Agency of Ministry of Education, Science, Research and Sport of Slovak Republic and Slovak Academy of Sciences (VEGA grant No. 1/0738/20).

The authors declare no conflict of interest.