All control and a subset of 230 PSP (PSP Series 1) participants had genotypes obtained in our prior study [8 (link)]. We genotyped all 973 LBD-NP and an additional 810 PSP (PSP Series 2) patients using the same methods.
DNA was extracted from blood using AutoGenFlexStar (AutoGen) and FlexiGene Chemistry (Qiagen) or brain using AutoGen 245T using standard protocols. Genotyping was performed using TaqMan assays (ABI3_rs616338, C_2270073_20; PLCG2_rs72824905, C_97909430_10) following manufacturers’ protocol, using a QuantStudio 7 Flex Detection System with a 384-Well Block Module (Applied Biosystems, Foster City, CA).
All minor allele carriers (ABI3_rs616338-T, PLCG2_rs72824905-G) were confirmed using Sanger Sequencing. Polymerase chain reaction (PCR) primers with the following sequences were used to amplify and sequence the genomic region flanking the mutations: ABI3 5′-CTTCCTGCTCGCACCCGAC-3′, 5′-CTAATGCAGCATCCCCAACT-207 3′, PLCG2 5′- CCATAAATGAGGGCTCTCAG-3′, 5′-CATACCCACCTCACCCTTGT-3′. PCR products were purified using the Agencourt AMPure protocol (Beckman Coulter, CA) and sequenced using a Big Dye Terminator v3.1 Cycle Sequencing Kit (Applied Biosystems). Sequencing reactions were purified using Agencourt CleanSEQ (Beckman Coulter, CA) and run on an ABI33730xl Genetic Analyzer (Applied Biosystems). Sequences were analyzed using Sequencher 4.8 (Gene Codes Corporation, MI).
DNA was extracted from blood using AutoGenFlexStar (AutoGen) and FlexiGene Chemistry (Qiagen) or brain using AutoGen 245T using standard protocols. Genotyping was performed using TaqMan assays (ABI3_rs616338, C_2270073_20; PLCG2_rs72824905, C_97909430_10) following manufacturers’ protocol, using a QuantStudio 7 Flex Detection System with a 384-Well Block Module (Applied Biosystems, Foster City, CA).
All minor allele carriers (ABI3_rs616338-T, PLCG2_rs72824905-G) were confirmed using Sanger Sequencing. Polymerase chain reaction (PCR) primers with the following sequences were used to amplify and sequence the genomic region flanking the mutations: ABI3 5′-CTTCCTGCTCGCACCCGAC-3′, 5′-CTAATGCAGCATCCCCAACT-207 3′, PLCG2 5′- CCATAAATGAGGGCTCTCAG-3′, 5′-CATACCCACCTCACCCTTGT-3′. PCR products were purified using the Agencourt AMPure protocol (Beckman Coulter, CA) and sequenced using a Big Dye Terminator v3.1 Cycle Sequencing Kit (Applied Biosystems). Sequencing reactions were purified using Agencourt CleanSEQ (Beckman Coulter, CA) and run on an ABI33730xl Genetic Analyzer (Applied Biosystems). Sequences were analyzed using Sequencher 4.8 (Gene Codes Corporation, MI).
Free full text: Click here
