A rare loss-of-function variant of ADAM17 is associated with late-onset familial Alzheimer disease

Abstract

Common variants of about 20 genes contributing to AD risk have so far been identified through genome-wide association studies (GWAS). However, there is still a large proportion of heritability that might be explained by rare but functionally important variants. One of the so far identified genes with rare AD causing variants is ADAM10. Using whole-genome sequencing we now identified a single rare nonsynonymous variant (SNV) rs142946965 [p.R215I] in ADAM17 co-segregating with an autosomal-dominant pattern of late-onset AD in one family. Subsequent genotyping and analysis of available whole-exome sequencing data of additional case/control samples from Germany, UK, and USA identified five variant carriers among AD patients only. The mutation inhibits pro-protein cleavage and the formation of the active enzyme, thus leading to loss-of-function of ADAM17 alpha-secretase. Further, we identified a strong negative correlation between ADAM17 and APP gene expression in human brain and present in vitro evidence that ADAM17 negatively controls the expression of APP. As a consequence, p.R215I mutation of ADAM17 leads to elevated Aß formation in vitro. Together our data supports a causative association of the identified ADAM17 variant in the pathogenesis of AD.

Fig. 1

Pedigree of AD family. The affected and unaffected individuals are filled in gray and white, respectively. The APOE status is given beneath individual identifiers. Carriers of the heterozygous variant (rs142946965) are indicated as C/A and carriers of the wild-type ADAM17 version are indicated as C/C

Fig. 2

The R215I variant affects maturation of ADAM17. a Western blot showing ADAM17 (A17) protein expression in SH-SY5Y cells, over-expressing wild-type (wt) ADAM17, a dominant-negative form (E406A), or the newly identified AD variant of ADAM17 (R215I). The parental cell line was analyzed as control, actin-beta was analyzed as loading control (Actin). b, c Quantification of pro-ADAM17 signals shows significant upregulation in cell lines over-expressing ADAM17 variants (n = 8; p(wt) = 0.0057; p(E406A) = 0.0009; p(R215I) = 0.0014), respectively, as compared to the parental cell line (control). Quantification of mature ADAM17 signals only shows significant upregulation in cell lines over-expressing wild-type ADAM17 (n = 8; p(wt) = 0.0092), as compared to the parental cell line (control). Asterisks indicate significance. Error bars indicate mean with SD

Fig. 3

APP expression is downregulated by ADAM17. a Western blot showing total APP protein expression in lysates of cells over-expressing APP and respective variants of ADAM17 (A17). b Quantification of total APP protein signals shows significant downregulation in cells over-expressing wild-type (wt) ADAM17 and APP as compared to cells only expressing APP (APP control). No significant change was observed in cells expressing ADAM17 E406A and R215I variants, respectively, as compared to APP control cells. Rescue of APP expression was observed in A17 wt cells after treatment with ADAM inhibitor TAPI-0. c APP gene expression is significantly downregulated in cells over-expressing A17 wt and APP as compared to APP control cells (n = 9, p(wt) = 0.001). Rescue of APP expression was observed in A17 wt cells after treatment with ADAM inhibitor TAPI-0 (n = 3). APP was also significantly downregulated in the parental cell line that did not over-express APP (n = 9, p = 0.0341). d ELISA of Aß₄₀ or e Aß₄₂ in media of SH-SY5Y cells over-expressing APP shows significant downregulation of both Aß forms in A17 (wt), (p(Aß40) < 0.0001; p(Aß42)) = 0.0007; n = 4) cells as compared to APP control cells. For b to e: Asterisks indicate significance. Error bars indicate mean with SD

Fig. 4

APP expression is negatively correlated with ADAM17 expression in human brain. Average log₂ of ADAM17 gene expression against average log₂ of APP gene expression was plotted. a NBB sample set; b MUC sample set