Additional file 1: of TMEM106B haplotypes have distinct gene expression patterns in aged brain

Table S1. Tissue samples available and selected for inclusion in this study. Table S2. DEGS in TCX. Positive fold change represents higher gene expression in SS than TT. Negative fold change represents lower gene expression in SS than TT. Table S3. DEGS in CER. Positive fold change represents higher gene expression in SS than TT. Negative fold change represents lower gene expression in SS than TT. Table S4. Overlapping genes between TCX and CER based on top 500 genes with |FC| ≥ 1.2 ranked by unadjusted p value. Table S5. Enrichment of modules for their respective DEG signatures. Table S6. Significant modules identified in the TCX and CER matched cases. Table S7. Significant modules identified in separate disease groups in TCX and CER. Table S8. Significant modules identified in the TCX and CER controls. (DOCX 54 kb

Additional file 1: Table S1. of Genome-wide pleiotropy analysis of neuropathological traits related to Alzheimer’s disease

Sample characteristics. Table S2. Association P values of Alzheimer disease loci previously established by GWAS in univariate and pleiotropy association tests of neuropathological features. Table S3. Association of cis-eQTL for HDAC9 in the Mayo Clinic brain expression genome-wide association study (eGWAS). rs79524815 was not available in the Mayo Clinic brain eGWAS, so proxy SNPs that are in LD (D′ > 0.90) with rs79524815 were used for the eQTL test with HDAC9 expression. Table S4. Association of expression of SNPs for TRAPPC12-AS1 and ADI1 with neuropathological traits and gene expression in the GTEx portal database. Table S5. Association results from the trivariate pleiotropy model of neuritic plaque (NP), neurofibrillary tangles (NFT), and cerebral amyloid angiopathy (CAA) for study-wide significant SNPs in the bivariate pleiotropy model. Table S6. Results of differential gene expression analysis by brain region among AD cases and controls for AD loci previously established by GWAS in RNA-Seq and microarray analysis. Figure S1. Quantile-quantile plots of observed (y-axis) vs. expected (x-axis) P values of all SNPs (black dots) and after excluding SNPs in APOE region (blue dots) for the pleiotropy analysis of (a) NP and NFT, (b) NP and CAA, and (c) NFT and CAA using the O’Brien method [10]. Figure S2. Manhattan plots showing genome-wide pleiotropy analyses of (a) NP and NFT, (b) NP and CAA, and (c) NFT and CAA using the O’Brien method [10]. Red dashed line represents the genome-wide significance threshold of P < 5.0 × 10−−8. Loci achieving genome-wide significance are highlighted in red, and known AD genes that attained at least a moderate significance level (P < 10−−4) are highlighted in gold. Figure S3. Regional association plots of genes, including TRAPPC12, TRAPPC12-AS1, and ADI1, on chromosome 2 from the joint model of NFT and CAA. Figure S4. Genome-wide trivariate pleiotropy analysis of NP, NFT, and CAA. (a) Quantile-quantile plot. (b) Manhattan plot. (DOCX 1028 kb

LRRTM3 Interacts with APP and BACE1 and Has Variants Associating with Late-Onset Alzheimer’s Disease (LOAD)

<div><p>Leucine rich repeat transmembrane protein 3 (LRRTM3) is member of a synaptic protein family. <i>LRRTM3</i> is a nested gene within α-T catenin (<i>CTNNA3</i>) and resides at the linkage peak for late-onset Alzheimer’s disease (LOAD) risk and plasma amyloid β (Aβ) levels. <i>In-vitro</i> knock-down of <i>LRRTM3</i> was previously shown to decrease secreted Aβ, although the mechanism of this is unclear. In SH-SY5Y cells overexpressing APP and transiently transfected with LRRTM3 alone or with BACE1, we showed that LRRTM3 co-localizes with both APP and BACE1 in early endosomes, where BACE1 processing of APP occurs. Additionally, LRRTM3 co-localizes with APP in primary neuronal cultures from Tg2576 mice transduced with LRRTM3-expressing adeno-associated virus. Moreover, LRRTM3 co-immunoprecipitates with both endogenous APP and overexpressed BACE1, in HEK293T cells transfected with LRRTM3. SH-SY5Y cells with knock-down of <i>LRRTM3</i> had lower <i>BACE1</i> and higher <i>CTNNA3</i> mRNA levels, but no change in <i>APP</i>. Brain mRNA levels of <i>LRRTM3</i> showed significant correlations with <i>BACE1</i>, <i>CTNNA3</i> and <i>APP</i> in ∼400 humans, but not in <i>LRRTM3</i> knock-out mice. Finally, we assessed 69 single nucleotide polymorphisms (SNPs) within and flanking <i>LRRTM3</i> in 1,567 LOADs and 2,082 controls and identified 8 SNPs within a linkage disequilibrium block encompassing 5′UTR-Intron 1 of <i>LRRTM3</i> that formed multilocus genotypes (MLG) with suggestive global association with LOAD risk (p = 0.06), and significant individual MLGs. These 8 SNPs were genotyped in an independent series (1,258 LOADs and 718 controls) and had significant global and individual MLG associations in the combined dataset (p = 0.02–0.05). Collectively, these results suggest that protein interactions between LRRTM3, APP and BACE1, as well as complex associations between mRNA levels of <i>LRRTM3, CTNNA3, APP</i> and <i>BACE1</i> in humans might influence APP metabolism and ultimately risk of AD.</p></div

Co-localization of LRRTM3 and a) APP in early endosomes, b) BACE1 in early endosomes, c) APP in primary neurons.

<p>For a and b, SH-SY5Y-APP695wt cells were transduced with baculovirus expressing fused early-endosomal protein Rab5a and GFP. These cells were transfected with LRRTM3-V5 (a and b) and also with BACE1-HA (b). Results of staining with GFP fluorescence indicative of Rab5a expression (early endosomes, green); anti-V5 (LRRTM3, red); and either CT20 in a (APP, magenta) or anti-HA in b (BACE1, magenta) are shown with overlay of the three stains in the last panels of a and b. Co-localization of APP, LRRTM3 and early endosomes is visualized as white punctate intracellular structures in the last panel of a(arrow and arrowhead). Co-localization of APP, BACE1 and early endosomes is visualized as white punctate intracellular structures in the last panel of b. (Magnification: ×63). For c, primary neuronal cultures from Tg2576 transgenic mice transduced with rAAV-LRRTM3-V5 were stained with anti-V5 (LRRTM3, green) and CT20 (APP, red). Overlay of the two stains reveals co-localization of LRRTM3 and APP visualized as yellow puncta in the cell body (thin arrow) and neuronal process (thick arrow). (Magnification: ×100).</p

Expression levels of genes in brains of <i>Lrrtm3</i> knock-out, heterozygote and wild type mice.

<p>Bar graphs depicting mean gene expression levels and error bars representing the standard deviations obtained from the averages of 3 animals per genotypic group where expression levels from each mouse brain is assessed in quadruplicate. Expression values are obtained by the delta Ct method, where geometric mean of HPRT and GAPDH is utilized as the control gene expression values. Average expression values (2?(-delta Ct))x100 were plotted on the y-axis. The three mouse genotypic groups are color-coded as shown in the inset. The genes with expression level measurements are shown in groups, with gene names depicted on the x-axis.</p

Relative expression levels of genes in H4 cells treated with three anti-<i>LRRTM3</i> and a control siRNA.

<p>Bar graphs depicting mean relative gene expression levels and error bars representing the standard deviations obtained from the averages of 2–6 experiments where each experiment is assessed in quadruplicate. Relative expression values are obtained by the delta delta Ct method, where HPRT is utilized as the control gene (delta Ct) and all results are normalized to one of the control wells (delta delta Ct). Relative expression values (2?(-delta delta Ct)) are plotted on the y-axis. The different siRNA treatment groups are color-coded as shown in the inset. The genes with expression level measurements are shown in groups, with gene names depicted on the x-axis.</p

Human brain expression correlations between levels of <i>LRRTM3</i>, and <i>CTNNA3</i>, <i>BACE1</i> or <i>APP</i>.

<p>Multivariable linear regression analyses were conducted while controlling for technical variables (plate, RIN), biological variables (diagnosis, age, sex, APOE4 dose) and variables accounting for cell loss, gliosis and vascularity by including as covariates expression levels of genes highly expressed in neurons (<i>ENO2</i>), astrocytes (<i>GFAP</i>), oligodendrocytes (<i>OLIG2</i>), microglia (<i>CD68</i>) and endothelial cell (<i>CD34</i>). The brain levels of <i>CTNNA3</i>, <i>BACE1</i> or <i>APP</i> were used as the outcome variable in a model, which included the above covariates and <i>LRRTM3</i> brain expression levels. Gene expression levels were detected in both the temporal cortex and cerebellum, for which results are shown separately. The significance (p), effect size (Beta) and 95% confidence interval of the effect size (95%CI) of <i>LRRTM3</i> expression for each of the tested genes are shown. Significant results are highlighted. Negative beta reflects an inverse relationship and positive correlation have a positive beta.</p

Multilocus genotype (MLG) analysis results.

<p>Logistic regression analysis with MLGs of 8 SNPs from Haplotype Block 1 are done controlling for age, sex, APOE4 dosage and series. Results from Cohorts 1, 2, the USA only cohorts and combined Cohorts 1+2 analyses are shown. CON = control and AD = Alzheimer's disease numbers and percentages for each MLG are shown. P = P values, OR = odds ratio, L95 = lower 95% and U95 = upper 95% confidence interval of OR for each MLG is shown for each analysis. REF = the most common MLG is used as the reference genotype. NA = results of MLGs where one group has 0 subjects or where there are less than 10 subjects in total are not available. MLGs with p<0.2 are bolded. Global p value of association for all MLGs are also shown. We note that while none of the individual MLGs would be significant after correcting for 31 tested MLGs, the global MLG associations do not require such a correction, so global p<0.05 is statistically significant. MLGs with total subject counts <10 in the combined Cohorts 1+2 are grouped into the MLG-rare group.</p

Validation of top cerebellar <i>cis</i>SNP/transcript associations in the temporal cortex.

<p>Of the 2,980 top <i>cis</i>SNP/transcript associations, 2,685 existed in the temporal cortex replication study. Some of these top associations are shown. Only one <i>cis</i>SNP/transcript pair is selected for depiction. The chromosome (CHR), SNP, Probe, Gene Symbol (SYMBOL) of these associations are depicted. The uncorrected (P), genome-wide (P_Bonf) and study-wide Bonferroni-corrected (P_Bonf-study) P values, Beta coefficient of association are shown for the combined (All) analyses in the cerebellar eGWAS and the temporal cortex replication study. Regression coefficients are based on the SNP minor allele using an additive model.</p

Variance of cerebellar probe expression levels due to technical, biological, and <i>cis</i>SNP effects.

<p>Results from some of the top probes are depicted. Only one probe is selected per gene for depiction. R2technical = variance due to technical variables only (i.e. plates, RIN). addR2covariates = added proportion of variance due to biological covariates (i.e. age, sex, ApoE4 dose), adjR2covariates = addR2covariates adjusted for technical variance, addR2best-SNP = proportion of variance due to the best <i>cis</i>SNP, adjR2best-SNP = addR2best-SNP adjusted for technical variance.</p