TREM2 Gene Compound Heterozygosity in Neurodegenerative Disorders

Abstract

Background:

Homozygous variants of the TREM2 and TYROBP genes have been shown to be causative for multiple bone cysts and neurodegeneration leading to progressive dementia (NHD, Nasu-Hakola disease).

Objective:

To determine if biallelic variants of these genes and/or oligogenic inheritance could be responsible for a wider spectrum of neurodegenerative conditions.

Methods:

We analyzed 52 genes associated with neurodegenerative disorders using targeted next generation sequencing in a selected group of 29 patients (n = 14 Alzheimer’s disease, n = 8 frontotemporal dementia, n = 7 amyotrophic lateral sclerosis) carrying diverse already determined rare variants in exon 2 of TREM2. Molecular modeling was used to get an insight into the potential effects of the mutation.

Results:

We identified a novel mutation c.401_406delinsTCTAT; p.(Asp134Valfs*55) in exon 3 of TREM2 in an Alzheimer’s disease patient also carrying the p.Arg62His TREM2 variant. Molecular modeling revealed that the identified mutation prevents anchoring of the TREM2 protein in the membrane, leaving the core of the Ig-like domain intact.

Conclusion:

Our results expand the spectrum of neurodegenerative diseases, where the carriers of biallelic mutations in TREM2 have been described for Alzheimer’s disease, and highlight the impact of variant burden in other genes on phenotypic heterogeneity.

Keywords

Alzheimer’s disease amyotrophic lateral sclerosis behavioral variant of frontotemporal dementia compound heterozygosity variant burden

INTRODUCTION

A common feature of neurodegenerative diseases is a chronic inflammatory process in response to accumulating protein aggregates. Anti-inflammatory responses and suppression of the pro-inflammatory condition in microglia are promoted by triggering receptor expressed on myeloid cells 2 (TREM2; MiM#605086) [1]. TREM2 is mainly expressed by microglia in the brain and seems to be important for optimal microglial functioning by stimulating phagocytosis without causing tissue inflammation. The extracellular region of TREM2 includes a single immunoglobulin domain and a stalk region followed by a transmembrane domain and an intracellular domain without a signaling motif. An interaction with a downstream signaling adaptor, TYRO protein tyrosine kinase-binding protein (TYROBP; MiM#604142), is required for the transmission of the signal [2]. In physiological conditions membrane-bound TREM2 is cleaved within the stalk region by the disintegrin and metalloproteinase domain-containing protein 10 protein (ADAM10; MIM#602192), releasing soluble TREM2 (sTREM2) [3 –5]. Cerebrospinal fluid (CSF) levels of sTREM2 in patients with neurodegenerative conditions can be markedly higher or lower than in healthy subjects [6]. Several studies have shown that homozygous loss-of-function mutations in TREM2 or TYROBP genes cause multiple bone cysts and neurodegeneration leading to progressive dementia (NHD, Nasu-Hakola disease; MIM#221770) [7]. Additionally, some studies have shown that biallelic variants (homozygous or compound heterozygous) of TREM2 are causative for the behavioral variant of frontotemporal dementia (bvFTD; MIM#600274) without bone cyst [8 –14]. Patients with NHD present mental abnormalities typical for bvFTD [15]. Of note, TREM2 variants in the heterozygous state have been described both as risk and causative factors for other neurodegenerative disorders [3 , 16–19].

Taking all this into account, we decided to check whether biallelic variants of TREM2 and TYROBP genes can be found in patients with Alzheimer’s disease (AD; MIM#104300), frontotemporal dementia (FTD), and amyotrophic lateral sclerosis (ALS; MIM#105400). Besides the entire TREM2 and TYROBP genes we also determined the coding sequence variants of 50 other genes related to various neurodegenerative conditions putatively forming the genetic background of variable clinical phenotypes.

MATERIALS AND METHODS

Subjects

Twenty-nine patients (14 AD, 8 bvFTD, 7 ALS) with previously identified rare variants in exon 2 of TREM2 were included in the study (Table 1). This group was selected among 603 patients by Sanger sequencing [13]. All participants were unrelated Caucasians of Polish origin. The AD and FTD patients were recruited between 2003 and 2018 from the outpatient memory clinic of the MSWiA Hospital in Warsaw, and the ALS patients from the Department of Neurology of the Medical University of Warsaw. The clinical diagnosis was based on recommended criteria [20 –24]. All patients with AD regardless of age of onset manifested dominant episodic memory problems, without atypical presentations. Behavior and executive disturbances with initial sparing of memory and visuospatial functions were described in all bvFTD patients. The study has been approved by the Ethics Committee of the MSWiA Hospital in Warsaw (approval number 203/2020) in accordance with the principles of the Helsinki Declaration of 1975. Written informed consent has been obtained from all individuals or their primary caregivers.

Table 1

Characteristics of the study group

ID	Diagnosis	sex	AO	APOE	TREM2 ex.2	Selected genetic variants	MAF
1	LOAD	M	73	3/3	p.Asp87Asn
2	LOAD	M	76	3/4	p.Asp87Asn	TREM2: c.482+54C>G	0.000127478
3	LOAD	F	71	4/4	p.Arg62His
4	LOAD	M	78	3/3	p.Arg62His	ADAM10: rs139857169	0.017
5	LOAD	M	79	4/4	p.Arg62His	TYROBP: rs111477177	0.03
6	LOAD	F	79	3/4	p.Arg62His
7	LOAD	M	82	3/4	p.Arg62His
8	LOAD	F	69	3/3	p.Arg47His
9	EOAD	F	50	4/4	p.Asp87Asn
10	EOAD	F	54	3/3	p.Arg62His	TYROBP: rs59399698	0.024
11	EOAD	F	55	2/4	p.Arg62His	TYROBP: rs59399698	0.024
12	EOAD	M	57	3/3	p.Arg62His	APP: p.V642I	Pathogenic
13	EOAD	F	63	3/4	p.Arg62His	TREM2: p.(Asp134Valfs*55)	Novel variant
14	EOAD	F	60	3/4	p.Arg62Cys
15	bvFTD	M	66	3/3	p.Asp87Asn / p.Asp87Asn
16	bvFTD	M	52	2/3	p.Arg62His
17	bvFTD	F	71	3/4	p.Arg62His	TYROBP: rs59399698†	0.024
18	bvFTD	F	66	4/4	p.Arg47His	TYROBP: rs59399698	0.024
19	bvFTD	F	54	3/4	p.Arg62His	TYROBP: rs111477177	0.03
						MAPT: p.G55R	Pathogenic
						SORL1: rs545522170	0.0001
						SORL1: rs138580875	0.0004
20	bvFTD	M	65	2/3	p.Arg62His
21	bvFTD	F	60	3/3	p.Arg62Cys / p.Thr66Met	ADAM10: rs139857169	0.017
22	bvFTD	F	51	3/3	p.Arg62Cys / p.Thr66Met	SORL1: rs117260922	0.0212
23	ALS	F	33	3/3	p.Asp87Asn	APP: rs140304729	0.0027
						ADAM10: rs139857169	0.017
						SORL1: rs62622819	0.0063
24	ALS	M	31	3/3	p.Asp87Asn	ADAM10: rs139857169	0.017
						SORL1: rs2298813	0.0518
25	ALS	F	49	3/3	p.Asp87Asn
26	ALS	M	62	3/3	p.Arg62His	SORL1: rs138438079	0.0006
27	ALS	F	49	3/3	p.Arg62His
28	ALS	F	54	3/3	p.Arg62His
29	ALS	M	60	2/3	c.41-2_3insA	TYROBP: rs111477177	0.03
						SORL1: rs117260922	0.0212
						SORL1: rs146903951	0.0111

AO, age of onset; LOAD, late onset AD; EOAD, early onset AD; †homozygosity; APOE, apolipoprotein E; MAF, Minor allele frequency in the non-Finnish European population according to The GnomAD; TYROBP RefSeq NM_001173514; ADAM10 RefSeq NM_001110.

Genetic analysis

DNA was extracted from venous blood using a standard salting out method. Genetic analysis was performed using targeted next generation sequencing. Genomic DNA was captured using SureSelect Custom DNA Target Enrichment Probes (Agilent Technologies, Santa Clara, CA, USA) and paired-end 150-nt sequencing was performed on an Illumina HiSeq4000 platform (Illumina, San Diego, CA, USA). FASTQ files were generated from the sequencing platform via the Illumina pipeline. The quality of raw reads was analyzed with the fastQC software v. 0.11.8.

Paired-end reads from FASTQ files were aligned to the human reference genome GRCh38 with the BWA-MEM software v. 0.7.17-r1188. Duplicates were marked with Samblaster v. 0.1.24. GATK BaseRecalibrator v. 4.1.3.0 was used to adjust base qualities. Variants were called with GATK HaplotypeCaller v. 4.1.3.0 (with the ‘-ERC GVCF’ option). User-defined intervals were used. Single sample genotyping was performed with GATK GenotypeGVCF v. 4.1.3.0 (with default parameters). Annovar was used for initial variant annotation.

The Target Seq gene panel contained the entire TREM2 and TYROBP gene sequences along with UTR sequences and further regulatory regions, as well as the coding sequences of 50 genes associated with AD, FTD, or ALS (ABCA7, ADAM10, ALS2, ANG, APOE, APP, ATXN2, BACH1, BIN1, C21orf2, C9orf72, CHCHD10, CHMP2B, CLU, CR1, CSF1R, DCTN1, FIG4, FUS, GRN, HNRNPA1, ITM2B, KIF5A, MAPT, MATR3, MYRF, NOTCH3, OPTN, PFN1, PICALM, PRNP, PSEN1, PSEN2, RIN3, SERPINI1, SETX, SIGMAR1, SOD1, SORL1, SPG11, SQSTM1, TARDBP, TBK1, TMEM106B, TOMM40, TTR, TUBA4A, UBQLN2, VAPB, VCP).

All studied patients were screened for the presence of the hexanucleotide repeat expansion in C9orf72 using the repeat primed PCR method as previously described [25].

Sanger sequencing (ABI 3130 DNA analyzer, Applied Biosystems, Foster City, CA) was used to confirm a novel TREM2 mutation. Data were analyzed using SeqScape Software version 2.6 (Applied Biosystems).

The sequence of the mutated allele (TREM2 insdel) was confirmed by digesting PCR products containing TREM2 exon 3 with RseI enzyme (Thermo Scientific, Waltham, MA) followed by their amplification and sequencing. A fragment of the chromatogram presenting the novel variant is included in the Supplementary Material (Supplementary Figure 1).

The cis or trans position of biallelic mutations (p.Arg62His / p.(Asp134Valfs*55)) in the carrier was established using digestion with AasI enzyme (Thermo Scientific, Waltham, MA) followed by sequencing according to the procedure described in Supplementary Figure 2. Compliance with HGVS nomenclature was verified using Mutalyzer program [26] (https://mutalyzer.nl/name-checker?description=NM_018965.4%3Ac.401_406delinsTCTAT).

Modeling of the effects of the frameshift mutation on the properties of TREM2

The putative properties of WT (wild-type) and mutated TREM2 proteins were predicted with the Quick2D tool implemented in the Tubingen Bioinformatics Toolkit. In particular, transmembrane regions were predicted with TMHMM and potential signal peptides with SIGNALP. Structural modeling of WT and mutant TREM2 was performed with I-TASSER. The I-TASSER models were subsequently confirmed by additional modeling with the Rosetta de novo fragment assembly method. To avoid biasing the predictions toward highly similar homologs from the Protein Data Bank, the maximum sequence identity cutoff was set to 30% in the I-TASSER runs. Similarly, in the fragment selection procedure done with the Rosetta webserver, the inclusion of homologous sequences was turned off. In total, four simulations with I-TASSER were performed: WT Ig-like domain (residues 19–130), WT Ig-like domain and the C-terminal disordered region (residues 19–171), mutant Ig-like domain (residues 19–143), and the mutant Ig-like domain and the C-terminal disordered region resulting from the frame-shift (residues 19–187). Confirmatory Rosetta runs were performed with the WT and mutant Ig-like domains (residues 21–130 and 21–143, respectively).

RESULTS

In a group of 29 patients (14 AD, 8 bvFTD, 7 ALS) with previously identified variants in exon 2 of TREM2 we sequenced a panel of 52 genes with known causative mutations or risk variants linked to neurodegenerative disorders.

A Target Seq analysis confirmed heterozygous causative mutation in the amyloid precursor protein (APP; MiM#104760) (RefSeq NM_201414: c.1924g>a; p.Val642Ile) in one patient with AD and in the microtubule-associated protein TAU (MAPT; MiM#1571450) (RefSeq NM_001123066: c.163g>a; p.Gly55Arg) in one patient with FTD [27]. No other known causative variants in the 52 examined genes associated with neurodegenerative diseases were identified in the other 27 patients.

TREM2 frameshift variant in an AD patient

We found a novel insdel mutation in exon 3 of the TREM2 gene (RefSeq NM_018965.4: c.401_406delinsTCTAT; p.(Asp134Valfs*55)) comprising a simultaneous deletion of six nucleotides (ATCACC) and insertion of five others (TCTAT) and resulting in a frame shift and introduction of a premature STOP codon. This variant is absent in a group of 943 unrelated Polish genomes, sequenced in the frame of Thousand Polish Genomes project (https://1000polishgenomes.com) [28]. We have not found this variant in NGS data from our previous studies of 380 persons without neurodegenerative disorders.

The proband carried also the p.Arg62His TREM2 variant.

Case report of p.(Asp134Valfs*55) TREM2 carrier: 64-year-old woman with a history of anxiety disorder, hypertension, hypercholesterolemia, bronchial asthma, spine degeneration, and smoking was admitted to the Department of Neurology due to her cognitive impairment. The patient (and her family) complained of memory problems for about a year, with a slow progressing course, difficulties in finding words, misallocations of objects, loss of interest, problems with spatial orientation, circadian arrhythmias, and a deterioration in functioning in everyday life. The patient had a high school degree, was an economist. No family history of dementia was reported.

Neuropsychological assessment revealed dominant episodic memory disorders in verbal and non-verbal modalities. Attentional, executive, and working memory were also impaired, along with poor naming, and visuoconstructional and praxis deficits. A slight decline in verbal fluency and abstractive thinking with concreteness was found. The cognitive profile suggested mild dementia with dominant episodic memory impairment, most probably due to AD. At the examination, no neurological signs were observed. Mini-Mental Score Examination (MMSE) was 22. Clinical Dementia Rating (CDR) was scored 1 and the patient required help. Brain scans pointed to the presence of general atrophy without focal changes, with spared hippocampal areas. CSF biomarkers for AD were determined (amyloid-β= 416.7 pg/ml; total tau >1200 pg/ml; and phospho tau = 165.9 pg/ml; cut off values: 610 pg/ml; 277 pg/ml; 55 pg/ml, respectively) [29]. The patient was coming for her annual visits accompanied by family, reporting constant progression, with typical AD course. However, since she developed more advanced stages of dementia, the contact with the patient has been lost. The family decided to switch the facility to closer to their residence, as the trip and the doctors’ appointments were too much burden for the patient.

To get an insight into the potential effects of the discovered frameshift on the TREM2 protein we performed several bioinformatic analyses. The predicted structure of the mutated protein is included in supplementary material (Supplementary Figure 3). We found that the frameshift disrupts both the transmembrane region anchoring the protein in the membrane and the ADAM10 protease cleavage site. At the same time, the frameshift creates a new sequence C-terminal to the Ig-like domain predicted to contain an additional short beta-strand. These observations prompted us to investigate potential structural effects of the mutation, in particular, whether the putative beta-strand could interfere with the structure of the Ig-like domain. To this end, we modeled wild-type and mutant TREM2 protein and also the Ig-like domain alone using I-TASSER (the obtained models were further confirmed with Rosetta). In the wild-type models, the Ig-like domain closely resembled the experimental structure (PDB code 6B8O), confirming the adequacy of both methods. In the mutant models, the sequence resulting from the frame-shift is largely unstructured and does not interfere with the Ig-like domain which, as in the case of the WT models, is similar to the experimental structure. This suggests that the identified mutation most likely leaves the core of the Ig-like domain intact and affects only the C-terminal region, preventing membrane anchoring of the mutated protein. It is therefore tempting to speculate that this TREM2 variant may constitute a functional analog of sTREM2.

Variant burden in genes associated with neurodegenerative conditions

Our analysis also revealed a known TREM2 intron variant (RefSeq NM_018965.4: c.482+54C>G; rs905993798, MAF = 0.00012 in GnomAD) in a 76-year-old AD patient carrying also the p.Asp87Asn TREM2 mutation. An analysis with Human Splicing Finder (http://www.umd.be/HSF/) [30] showed that the c.482+54C>G variant likely disturbs mRNA splicing. No other rare, infrequent or polymorphic variants in the entire intronic and coding sequence of TREM2 were found in the remaining 27 patients. Also, the TYROBP gene sequence did not reveal any known causative variants. Numerous rare variants (MAF ≤0.05) of unknown consequences were identified in the coding and UTR sequences of the 50 other analyzed genes associated with neurodegenerative conditions. All relevant data are presented in Supplementary Data File 1. Potentially interesting variants, more frequent in our study group comparing to The Genome Aggregation Database (https://gnomad.broadinstitute.org/) [31], are listed in Table 1.

DISCUSSION

Mounting evidence indicates a role of TREM2 in the development of inflammatory neurodegenerative diseases [1 , 33]. So far, a number of rare variants in the TREM2 gene have been described, mainly in the second exon encoding the ligand-binding N-terminal domain. It is proposed that risk factors for AD (e.g., p.Arg47His, p.Arg62His, and p.Asp87Asn variants) influence ligand binding [5 , 34], while the NHD-associated variants considered causative lead to protein misfolding followed by impaired maturation, cell surface transport and proteolytic processing and resulting in a severe biochemical and clinical phenotype [5 , 35–37].

According to molecular modeling, the novel variant p.(Asp134Valfs*55) identified by us in the transmembrane domain of TREM2 completely prevents the anchoring of the receptor in the cell membrane and as a result disrupts signal transduction, resembling functionally the NHD-associated variants. As the core of the Ig-like domain seems intact, the novel variant most likely acts as a functional analog of soluble TREM2 (sTREM2).

Growing evidence suggests an ambiguous biological function of sTREM2 in microglial dynamics [38, 39]. It plays a protective role against amyloid-β assembly and macrophage apoptosis [40 –42], but on the other hand it also participates in an ongoing inflammatory process [5, 43]. The sTREM2 level changes dynamically during the progression of an inflammatory neurodegenerative disorder, peaking at early symptomatic stages of the disease and then declining [44]. It seems that not the absolute level of sTREM2 but rather its changes are a key element of pathological processes. For instance, p.Arg47His carriers present significantly higher, while carriers of the NHD-associated variants significantly lower CSF sTREM2 levels than controls [39, 44]. The p. His157Tyr variant enhancing cleavage of TREM2 lowers TREM2 - dependent phagocytosis and simultaneously increases sTREM level [45], which features are likely shared by our novel p.(Asp134Valfs*55) variant. In the Han Chinese population p. His157Tyr is defined as a significant risk factor for AD [46]. This variant is absent in neurologically healthy Caucasians but has been found in AD and FTD Caucasian patients [11 , 47]. All in all, it seems that variants altering the sTREM2 level can affect the disease risk/course through distinct pathogenic mechanisms besides the ligand-dependent one [42].

Mutation of both alleles of TREM2 seems to be associated with a severe course and early onset of the disease, whereas a partial loss of function due to single allele damage contributes to a mild phenotype in a way probably depending on other genetic factors, as heterozygous variants are also found in healthy relatives of biallelic patients and in control groups [11 , 47–49]. To date, homozygosity or compound heterozygosity in the TREM2 gene have been reported only in NHD patients and in a few patients with bvFTD.

To the best of our knowledge, the biallelic TREM2 genotype identified by us represents the first case described in a patient with an AD phenotype. As we do not have access to the patient’s relatives, we are not able to analyze co-segregation of mutations in the family, nor to exclude de novo mutation, undoubtedly indicating the pathogenic effect of compound heterozygosity, we have identified.

Of note, the intronic mutation in TREM2 (c.482+54C>G) found here in another AD patient carrying also the p.Asp87Asn variant. This intronic variant might cause alternative splicing resulting in exon 3 deletion, as has been described previously in patients with the NHD phenotype and c.482+2T>C mutation [7, 50]. The extremely low frequency of the c.482+54C>G variant in the general population (MAF = 0.00012) seems to confirm its negative effect. We have no access to the patient’s tissue for mRNA analysis.

In addition, our study recorded a much higher frequency of variants in the TYROBP and ADAM10 genes in the test group than in general population (non-Finnish European population in gnomAD) (see Supplementary Material and Table 1) [31]. We also identified in both ALS and FTD patients a high burden of rare variants in the sortilin-related receptor gene (SORL1; MiM#602005) (see Supplementary Material and Table 1). SORL1 has recently been associated with and intensively studied in AD context, as it encodes a key protein involved in the processing and secretion of APP [51]. What is more, we found a rare variant (Europe MAF = 0.0013), rs140304729 (RefSeq NM_201414: c.1570g>a; p.Glu524Lys) in exon 12 of APP in an ALS patient, while numerous mutations in APP have been associated causally with AD. According to prediction programs (SIFT, PolyPhen, REVEL, MetaLR), this variant is causative (https://www.ensembl.org). It is widely accepted that genome variants other than the main causative mutation modify the phenotype or are co-causative. The high variant burden in numerous genes associated with neurodegenerative conditions can result in a more complex phenotype and explain phenotypic heterogeneity and clinical overlap among neurodegenerative disorder patients [52 –56].

In conclusion, finding an AD patient with TREM2 compound heterozygosity expands the spectrum of neurodegenerative diseases, where the carriers of biallelic mutations in TREM2 have been described. Moreover in the patients with single TREM2 variants, we have demonstrated the simultaneous presence of rare variants in other genes related to neurodegeneration.

Limitations of the study

We have no access to the patient’s tissue for further analysis of cellular mechanisms or even checking the mRNA level. Also due to the limited size of the study group, it was not possible to support our observations with statistical analyzes. However, our report may be considered as a part of a bigger global study on the role of rare variants in the pathogenesis of neurodegenerative disorder and phenotypic variability.

Footnotes

ACKNOWLEDGMENTS

The authors would like to thank all the patients and their families involved in this study.

The research was supported by The National Science Centre of Poland, grant MINIATURA-2 no. 2018/02/X/NZ5/01509. JL and SDH were supported by the First Team programme of the Foundation for Polish Science co-financed by the European Union under the European Regional Development Fund [POIR.04.04.00-00-5CF1/18-00 to SDH].

Authors’ disclosures available online ().

The supplementary material is available in the electronic version of this article: .

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