Study population
Clinical information, which included age, sex, diagnosis, and APOE ε4 allele genotype, and the genomic DNA of LOAD patients and normal cognitive function (NC) control participants were obtained from the National Center for Geriatrics and Gerontology (NCGG) Biobank. The NCGG Biobank is one of the facilities belonging to the National Center Biobank Network (NCBN; https://ncbiobank.org/en/home.php). It has been running since 2012. The participants were recruited from NCGG hospital, which is located in Obu city, and the other nearby medical institutes. LOAD was diagnosed at NCGG hospital according to the diagnostic criteria developed by the National Institute on Aging and the Alzheimer’s Association (NIA-AA). (Albert et al., 2011; McKhann et al., 2011) We also applied regional cohort samples of elderly adults (≥ 65 years) in Aichi prefecture, stored in the NCGG Biobank, as general Japanese population samples for the association study. The demographic features of the NCGG samples applied in the exome sequencing and genotyping are shown in Additional file 1: Tables S1–S3. In the first cohort of the association study of two variants, rs572750141 and rs531355933, we also included 7345 DNA samples recruited at RIKEN and public whole-genome sequence data of 3554 Japanese individuals (3.5KJPN, Integrative Japanese Genome Variation; https://ijgvd.megabank.tohoku.ac.jp/) from Tohoku Medical Megabank Organization (TMM).
For the second cohort of the association study of the two variants identified, we obtained 2180 LOAD cases and 2486 controls recruited from Niigata University (Additional file 1: Table S4); their demographics and the clinical criteria used for the diagnosis of AD are described in a previous report. (Miyashita et al., 2014) All individuals are Japanese and provided written informed consent, and the study was performed with the approval of the ethics review board at NCGG and of each institute.
Exome sequencing and variant calling
For whole-exome sequencing, we used two kinds of next-generation sequencers, a HiSeq 2500 (Illumina, San Diego, CA) and an IonProton (Thermo Fisher Scientific, Waltham, MA). Genomic DNA samples were quantified using the Quant-iT™ PicoGreen® dsDNA Assay Kit or Qubit dsDNA HS Assay Kit (Thermo Fisher Scientific).
For HiSeq, exome libraries were prepared with the Nextera Rapid Capture Expanded Exome Kit (Illumina) or SureSelect Human All Exon V6 (Agilent Technologies, Santa Clara, CA) according to the manufacturers’ protocols. Enriched exome libraries were analyzed by using an Agilent 4200 TapeStation (Agilent) or DNA Fragment Analyzer (Advanced Analytical, Ankeny, IA). The libraries were then sequenced on a HiSeq 2500 system. Paired-end reads of 125 nucleotides were sequenced by using the HiSeq PE Cluster Kit v4 cBot and HiSeq SBS Kit v4. Data processing was performed by using a Resequence/Exome analysis pipeline (Amelieff, Tokyo, Japan). All software in the pipeline was used with properly tuned default settings. Briefly, for use in this pipeline, raw data from the HiSeq system were converted to the FASTQ format with bcl2fastq (Illumina). At the beginning of the pipeline, the FASTQ-formatted files were cleaned up with QCleaner software (Amelieff). Then, sequence reads were mapped to the human genome (hg19) using a BWA algorithm (http://bio-bwa.sourceforge.net/). Duplicated reads were removed by applying Picard (http://broadinstitute.github.io/picard/). Variant calling for single-nucleotide variants (SNVs) and indels was performed with GATK (https://www.broadinstitute.org/gatk/), and variants were outputted in VCF format.
For the IonProton sequencer, exome libraries were prepared with the Ion TargetSeq Exome Enrichment Kit or Ion AmpliSeq Exome RDY Kit (Thermo Fisher Scientific) according to the manufacturer’s protocol. The enriched exome libraries were analyzed by real-time PCR with the GeneRead Library Quant Kit (QIAGEN, Hilden, Germany). Then, libraries were sequenced on an IonProton system, and variants were called by Torrent Suite Software. Variants were outputted in VCF format.
Annotation
Variants were annotated with dbSNP rs identifiers (rs ID, NCBI dbSNP build 147), allele frequencies, and Combined Annotation Dependent Depletion (CADD) scores (Kircher et al., 2014) by using the ANNOVAR program (http://annovar.openbioinformatics.org/). (Wang et al., 2010) For CADD scores, variants were annotated with CADD version 1.3 (cadd13). For allele frequencies in public databases, we used data from the 1000 Genomes Project (1000g2015aug), Exome Sequencing Project (esp6500siv2_all), and Exome Aggregation Consortium (exac03). We also annotated allele frequencies in the Japanese population by using the 2KJPN database (https://ijgvd.megabank.tohoku.ac.jp/) from TMM.
Gene expression data
We used gene expression data from the Genotype-Tissue Expression (GTEx) project (https://gtexportal.org/). Median reads per kilobase of exon per million mapped reads (RPKM) data of 13 brain regions from GTEx Analysis V6 were averaged for each gene and used for the variant-filtering step.
Primers and probes
All primers for PCR reactions, Sanger sequencing, and invader assays were synthesized commercially (Fasmac, Kanagawa, Japan). Primers were designed using the Primer3Plus program (http://primer3plus.com/cgi-bin/dev/primer3plus.cgi).
Sanger sequencing
PCR was performed using AmpliTaq Gold 360 DNA Polymerase (Thermo Fisher Scientific). Purified PCR fragments were sequenced using the BigDye Terminator v3.1 Cycle Sequencing Kit and an ABI 3100 Genetic Analyzer (Thermo Fisher Scientific).
Genotyping
We genotyped candidate variants using a multiplex PCR invader assay (Third Wave Technologies, Madison, WI) (Ohnishi et al., 2001) by means of a QuantStudio 7 Flex Real-Time PCR System (Thermo Fisher Scientific) for NCGG and Niigata samples or ABI7900HT Fast Real-Time PCR System (Applied Biosystems) for RIKEN samples. For part of the first cohort control for the two loci (rs572750141 and rs531355933), we also obtained the genotyped data from 1765 in-house whole-genome sequences at RIKEN and the whole-genome sequence data of 3554 Japanese individuals at TMM (3.5KJPN, Integrative Japanese Genome Variation; https://ijgvd.megabank.tohoku.ac.jp/) (Additional file 1: Table S3).
Construction of plasmid vectors
Each PCR product for wild-type and G186R variant SHARPIN was prepared by using complementary DNA synthesized based on mRNA extracted from the buffy coat of the patients analyzed in this study and cloned into pCMV-Myc vector. All inserted sequences were confirmed by Sanger sequencing.
Luciferase assay
HEK293 cells were transfected with the luciferase reporter plasmid pGL4.32[luc2P/NF-κB-RE/Hygro] (Promega, Madison, WI), and stably expressing cells were selected by hygromycin. Twenty-four hours before transfection, cells were plated on 96-well plates (1.5 × 104 cells/well). Transfection with plasmids was performed using FuGENE® HD Transfection Reagent (Promega). Twenty-four hours after transfection, cells were treated with tumor necrosis factor-α (TNF-α, 20 ng/ml) (Wako, Osaka, Japan) for 5 h. Then, luciferase activity was measured by using a Nano-Glo® Dual-Luciferase® Reporter Assay System (Promega). Each experiment was independently performed three times with five replicates of each sample.
Immunocytochemistry
HEK293 cells were seeded at a density of 2.0 × 104 cells/well on BioCoat™ Poly-D-Lysine 4-well Culture Slides (Corning, NY, USA), cultured for 24 h, and transfected with wild-type and G186R Myc-SHARPIN plasmids. Twenty-four hours after transfection, cells were fixed and incubated with Anti-Myc-tag mAb-Alexa Fluor® 488 (MBL, Nagoya, Japan) according to the manufacturer’s protocol. Then, the slides were mounted with SlowFade™ Diamond Antifade Mountant with DAPI (Thermo Fisher Scientific) and fluorescence images were obtained on a BIOREVO BZ-9000 fluorescence microscope (Keyence, Osaka, Japan).
Statistical analysis
All statistical analyses were performed using R software (version 3.2.4). For calculation of the odds ratio and 95% confidence interval, vcd package (version 1.4.3) was used in R. Meta-analyses of two cohort sets were performed by using the Mantel-Haenszel χ2 test. For luciferase assay experiments, a Student’s t-test was conducted to estimate the statistical difference in luciferase activity among cells transfected with mock (expressing myc), wild-type, and G186R plasmids.