TYROBP genetic variants in early-onset Alzheimer's disease

Abstract

We aimed to identify new candidate genes potentially involved in early-onset Alzheimer's disease (EOAD). Exome sequencing was conducted on 45 EOAD patients with either a family history of Alzheimer's disease (AD, <65 years) or an extremely early age at the onset (≤55 years) followed by multiple variant filtering according to different modes of inheritance. We identified 29 candidate genes potentially involved in EOAD, of which the gene TYROBP, previously implicated in AD, was selected for genetic and functional follow-up. Using 3 patient cohorts, we observed rare coding TYROBP variants in 9 out of 1110 EOAD patients, whereas no such variants were detected in 1826 controls (p = 0.0001), suggesting that at least some rare TYROBP variants might contribute to EOAD risk. Overexpression of the p.D50_L51ins14 TYROBP mutant led to a profound reduction of TREM2 expression, a well-established risk factor for AD. This is the first study supporting a role for genetic variation in TYROBP in EOAD, with in vitro support for a functional effect of the p.D50_L51ins14 TYROBP mutation on TREM2 expression.

Keywords: Alzheimer's disease; Burden test; Exome sequencing; TREM2; TYROBP.

Fig. 1. Molecular genetic analysis of TYROBP

(A) Panel A includes a chromatogram of several bases surrounding the p.G2E variant (red square) of patient A and an healthy subject. (B) Schematic representation of TYROBP protein along with its different domains and the rare coding variants identified in the overall EOAD cohort. A PCR was performed on the cDNA from patient A using a PCR primer set with a reverse primer specific for the mutant T allele of the p.V55L variant (NM_003332.3:c.163G>T) allowing us to specifically amplify the chromosome carrying the c.163T allele. This PCR fragment also spanned the sequence surrounding the p.G2E (NM_003332.3:c.5G>A) variant. Sanger sequencing of the PCR product showed the mutant A allele of the p.G2E variant suggesting that the variant is in cis with the p.V55L variant.

Fig. 2. Effect of TYROBP mutant p.D50_L51ins14 on TREM2 expression

(A) Co-expression of TYROBP mutant with TREM2 decreases the level of TREM2 protein expression. Western blot of HeLa cells transfected with TREM2-Myc in combination with wild-type TYROBP-eGFP (TYROBP WT-eGFP) or with the p.D50_L51ins14 (TYROBP mutant-eGFP) or a vector control eGFP. The levels of TYROBP and TREM2 were visualized using an eGFP or cMyc antibody, respectively. GAPDH was used as loading control. (B) Quantification of TYROBP and TREM2 protein levels in HeLa cells transfected as described in (A). Quantification is assessed relative to the TYROBP WT-eGFP transfection condition after normalization to GAPDH levels. (C) Quantification of mRNA levels in HeLa cells transfected as described in (A). **: p<0.01; ***: p<0.0001, Student t-test on n≥4 replicates.