Monoallelic TYROBP deletion is a novel risk factor for Alzheimer's disease

Abstract

Biallelic loss-of-function variants in TYROBP and TREM2 cause autosomal recessive presenile dementia with bone cysts known as Nasu-Hakola disease (NHD, alternatively polycystic lipomembranous osteodysplasia with sclerosing leukoencephalopathy, PLOSL). Some other TREM2 variants contribute to the risk of Alzheimer's disease (AD) and frontotemporal dementia, while deleterious TYROBP variants are globally extremely rare and their role in neurodegenerative diseases remains unclear. The population history of Finns has favored the enrichment of deleterious founder mutations, including a 5.2 kb deletion encompassing exons 1-4 of TYROBP and causing NHD in homozygous carriers. We used here a proxy marker to identify monoallelic TYROBP deletion carriers in the Finnish biobank study FinnGen combining genome and health registry data of 520,210 Finns. We show that monoallelic TYROBP deletion associates with an increased risk and earlier onset age of AD and dementia when compared to noncarriers. In addition, we present the first reported case of a monoallelic TYROBP deletion carrier with NHD-type bone cysts. Mechanistically, monoallelic TYROBP deletion leads to decreased levels of DAP12 protein (encoded by TYROBP) in myeloid cells. Using transcriptomic and proteomic analyses of human monocyte-derived microglia-like cells, we show that upon lipopolysaccharide stimulation monoallelic TYROBP deletion leads to the upregulation of the inflammatory response and downregulation of the unfolded protein response when compared to cells with two functional copies of TYROBP. Collectively, our findings indicate TYROBP deletion as a novel risk factor for AD and suggest specific pathways for therapeutic targeting.

Keywords: Alzheimer’s disease; DAP12; Genetics; Nasu-Hakola disease; Polycystic lipomembranous osteodysplasia with sclerosing leukoencephalopathy; TYROBP.

Fig. 1

The Finnish 5.2 kb TYROBP deletion. A WGS data show homozygous 5.2-kb deletion encompassing TYROBP exons 1–4 in a Finnish NHD patient. The horizontal lines indicate WGS reads spanning across the deletion. The deletion breakpoints are located within a 23-bp identical sequence (black, underlined). B Schematic of the TYROBP deletion founder haplotype. The boxes indicate haplotype blocks identified using microsatellite markers (grey bars) [13]. The grey shading indicates the haplotype region shared by all Finnish NHD patients, while the red shading indicates the 5.2-kb TYROBP deletion. Asterisks denote the haplotypes detected in the NHD patients in the current study. Purple lines indicate the SNVs identified in the current study within the shared haplotype region. C Regional allele frequency of rs1244787406-G in Finland. The black line separates the early settlement region along the southern and western coastlines from the late settlement region that was permanently inhabited from the sixteenth century onwards by internal migration (arrows) mainly from the current South Savo region in the southeastern Finland. The circles represent the birth places of the grandparents of the Finnish NHD patients according to [43]. The highest present day allele frequencies of the TYROBP deletion proxy marker rs1244787406-G are detected in the late settlement area and coincide with the previously reported regional enrichment

Fig. 2

Phenotype associations for the TYROBP deletion proxy marker rs1244787406-G. A Phenotype association study covering 2,489 endpoints indicates significantly increased risk for dementia and AD among carriers of rs1244787406 minor G allele. The dashed line represents genome-wide significance threshold P = 5 × 10⁻⁸. B-C Kaplan–Meier survival plots showing the proportion free from dementia (B) and AD (C) among rs1244787406-G carriers (purple line) and non-carriers (black line). Shading indicates 95% confidence intervals. X-axis indicates age at the first diagnosis for cases and age at the end of follow-up for controls. D Regional association plot of the TYROBP locus shows the negative log10-transformed P-values on the y axis for the endpoint ‘Dementia (including primary healthcare outpatient registry)’ derived from FinnGen. The vertical dashed line represents genome-wide significance threshold P = 5 × 10⁻⁸. Each dot represents an individual SNV, and the dot color represents LD with the LD reference variant (purple diamond). The dotted line in the upper panel delineates the area shown in the lower panel. The red vertical lines indicate the 5.2 kb TYROBP deletion break points while the shaded grey area represents the TYROBP deletion associated shared haplotype region in the lower panel

Fig. 3

Clinical imaging of a monoallelic TYROBP deletion carrier and an NHD patient. A-B Radiographs of wrists and ankles of the monoallelic TYROBP deletion carrier show small cystic-appearing translucencies in the lunate bones of the wrists and the left calcaneus and distal tibia of the ankle, as delineated by arrowheads (C) Post-traumatic and postoperative radiographs of the NHD patient’s right wrist over time span of 5 years. The bone structure is patchy already in the earlier radiograph (on the left), with cystic-appearing translucencies notably in the lunate and capitate. Five years later the primary findings are even more pronounced and all carpal bones as well as the distal radius and ulna present as abnormal (within the ellipse). D Radiograph of the NHD patient’s ankles shows numerous abnormal translucencies in the distal heads of the tibia and fibula, talar, calcaneal, and cuboid bones. E Magnetic resonance images (MRI) of the monoallelic TYROBP deletion carrier. No signal or structural pathologies are observable, and there is no evidence of marked atrophy on any of the contrast sets (FLAIR, T1- or T2-weighted, or susceptibility weighted images (SWI)). F Computed tomography (CT) and MRI images of the brain of the NHD patient show bilateral calcifications of the basal ganglia and frontal white matter as hyperdensities in the CT scan, denoted by purple arrowheads. Some punctate signal voids can be observed in the corresponding regions on MRI by SWI both at 3.0 T and at 1.5 T field strengths (arrows). FLAIR, T2- or T1-weighted images show no obvious signal pathology. Marked cortical and temporomesial atrophy is however present and shows rapid progression over time

Fig. 4

Effect of the Finnish 5.2-kb TYROBP deletion on AD-related Aβ pathology. A A representative image of frontal cortex biopsy immunostained for microglia (Iba- 1, brown) and Aβ (red). Scale bar 50 µm. B-D Number (B) and size (C) of Aβ plaques, and the number of Aβ plaque-associated microglia (D) in immunohistochemical images shown in (A); n = 1–5 individuals per genotype. E Aβ42 levels in the CSF; n = 1–4 individuals per genotype. Data are shown as mean ± SD. Independent samples T-test for T^CV/A³³ and T^CV/A³⁴. *: P < 0.05. T^CV, TYROBP common variant; T^del, monoallelic TYROBP deletion; A³³, APOE ε3ε3; A.³⁴, APOE ε3ε4

Fig. 5

Characterization of the MDMi cell model. A Study design to assess functional effects of the TYROBP deletion in MDMi cells. B Decreased DAP12 protein levels in the monocytes and MDMi of monoallelic TYROBP deletion carriers compared to cells from individuals homozygous for the TYROBP common variant. DAP12 protein levels were normalized to GAPDH levels in the same lysate. Monocytes: T^CV, n = 4; T^del, n = 2; MDMi: T^CV, n = 2; T^del, n = 3. C TYROBP RNA levels are decreased in monocytes and MDMi of monoallelic TYROBP deletion carriers compared to cells from individuals homozygous for TYROBP common variant, while no TYROBP RNA is detected in NHD patients homozygous for the TYROBP deletion. Monocytes: T^CV, n = 2; T^del, n = 2; NHD, n = 2; MDMi: T^CV, n = 8; T^del, n = 3; NHD, n = 2. D Principal component analysis reveals that MDMi and iPSC-derived microglia (iMG) group closely together according to their expression profiles, which were different from their respective precursor cells, monocytes and iPSCs. E–F GSEA analysis shows that microglial genes are upregulated (E) while monocyte genes are downregulated (F) in the MDMi cells compared to monocytes. G TREM2 is not expressed in monocytes and is upregulated during MDMi differentiation in all genotype groups. Monocytes: T^CV, n = 2; T^del, n = 2; NHD, n = 2; MDMi: T^CV, n = 8; T^del, n = 3; NHD, n = 2. Data in B, C, and G are shown as mean ± SD. Each data point represents one individual

Fig. 6

Increased inflammatory response and decreased unfolded protein response in LPS-treated MDMi cells with TYROBP deletion. A-C Volcano plots of differentially expressed genes and (D-F) proteins in the monoallelic TYROBP deletion carrier MDMi cells compared to controls upon untreated (A, D), myelin-treated (B, E) and lipopolysaccharide (LPS)-treated (C, F) conditions., T^CV, n = 3–12; T^del, n = 3. (G) Pathway enrichment of genes (left panel) and proteins (right panel) differentially expressed in the monoallelic TYROBP deletion carrier MDMi cells compared to TYROBP common variant MDMi cells. H-I Heat maps showing the top up- and downregulated targets in the UPR and MYC pathways in the monoallelic (H) and biallelic (I) TYROBP deletion carriers compared to controls. J-K Inflammatory response was assessed by measuring interleukin-6 (IL-6), IL-1β, IL-10 (J), and tumor necrosis factor α (TNFα; K) levels in the conditioned media of T^CV, T^del, and NHD MDMi upon 24 h LPS treatment. Each data point indicates one individual, an average of 1–3 replicate wells. T^CV, n = 3–4; T^del, n = 3–4; NHD, n = 1–2. All data in J are shown as mean ± SD. Independent samples T-test, *: P < 0.05. T^CV, TYROBP common variant; T^del, monoallelic TYROBP deletion; NHD, Nasu-Hakola disease