Multiplex snapshot technique

Genomic DNA from all subjects was extracted from whole blood samples using 0.5 M ethylene diamine tetraacetic acid (EDTA) as an anticoagulant, using the QiaAmp DNA Mini Kit (Qiagen, Hilden, Germany). The DNA samples were stored at −20°C. Genotyping of rs12134493 and rs2078371 in TSPAN2 was performed using the Multiplex SNaPshot technique (Applied Biosystems by Life Technologies, Foster City, CA, USA). PCR amplifications were performed in a final volume of 25 μl, containing 50 mM MgCl₂, 10 mM dNTP, 1 μM primers, and 5 units of Platinum Taq DNA polymerase. The PCR conditions used were 95°C denaturation for 2 min; followed by 33 cycles at 95°C for 20 s, 55°C for 20 s, and 72°C for 40 s; and a final extension step at 72°C for 5 min. The SNaPshot reaction was performed in a final volume of 5 μl (reaction mix 2.5 μl, PCR products 1.5 μl, and probe mix 1.0 μl). DNA sequencing was performed in a final volume of 10 μl containing 1 μl of SNaPshot purified product, 8.5 μl of deionized formamide, and 0.5 μl GeneScan-120 LIZ Size Standard, using an ABI PRISM 3730 DNA Sequencer (Applied Biosystems by Life Technologies). All sequence analyses were performed using GeneMapper4.0 DNA Sequencing Analysis software.

The HapMap database was adopted to search for single nucleotide polymorphisms (SNPs) in DYCN1H1 gene representing Han‐Chinese. The linkage disequilibrium (LD) was determined by Haploview software version 4.0 to screen candidate tag SNPs. Tap SNPs with minor allele frequency (MAF) > 0.01 and threshold of r² > 0.8 were further investigated. Ultimately, a total of nine tag SNPs in DYCN1H1 gene (rs1004903, rs10131545, rs10132469, rs11160668, rs1190605, rs1190606, rs12161908, rs2273440, and rs3818188) were selected.
The QIAGEN DNA extraction kit (Germany) was employed to acquire genomic DNA from peripheral blood samples. DNA samples were preserved in the medical refrigerator at −80℃ until being used. SNPs genotyping was performed using the Multiplex SNaPshot technique based on ABI fluorescence‐based assay discrimination method (Applied Biosystems, Foster City, CA). The SNaPshot information of DYNC1H1 gene was presented in Supplementary Table S1.

Venous blood specimens are collected directly from all PD patients and the healthy controls with ethylene diamine tetraacetic acid (EDTA) anticoagulant. Genomic DNA is extracted from the blood samples with the QIAamp DNA Mini Kit (Qiagen, Hilden, Germany) under the manufacturer's instructions and stored at −20°C. The five SNPs are genotyped by Multiplex Snapshot technique (Applied Biosystems by Life Technologies, Foster City, CA, USA). The primers are designed for each SNP locus using Primer 5 and listed in Table 2. Multiplex PCR reactions are performed to amplify target regions containing the selected SNPs. All products are analyzed by the ABI PRISM 3730 DNA Sequence, of which the sequence analyses are conducted by DNA Sequencing Analysis software, GeneMapper4.0. To confirm the results, 10% patients and 10% controls are randomly selected for Sanger sequencing approaches. The concordance rate for replicate approaches was 100%.