The SNCA gene expression dataset and analysis methods used here have been described previously49 (link). In brief, SNCA expression levels were assayed from 165 samples using quantitative real-time polymerase chain reaction. The Relative Standard Curve Method was used to transform the Ct values into quantity units. The base 10 logarithm of the SNCA expression values was used for all analyses, to ensure the normal distribution of data required by the statistical tests performed. SNP genotyping was performed as part of the US PD-GWAS Consortium meta-analysis replication sample50 (link). As described in the consortium study, the samples were genotyped at the Center for Inherited Disease Research (CIDR) using a custom Illumina genotyping array of 768 SNPs. Because the tested SNP dataset did not include the top reported PD-associated SNP rs356182 in the SNCA locus, we included rs356229 as proxy for this SNP (R2 = 0.62) in our analysis. After QC, genotyping and expression data was available for 86 cases and 41 controls for eQTL analysis. Expression models were analyzed including adjustment for disease status, sex, pH, age at death, as well as for the interaction between PMI and disease status and significance was assessed using a one sided test based on the a priori hypothesis of an association of the G-allele at rs356168 with increased SNCA expression.
Genotyping array
Manufactured by Illumina
The Genotyping array is a laboratory equipment used to analyze genetic variations within an individual's DNA. It allows for the simultaneous measurement of thousands of genetic markers across the genome.
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7 protocols using genotyping array
1
Examining SNCA Expression and Genotypes
2
Illumina SNP Genotyping Protocol
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SNP variants were called by technicians at the Illumina FastTrack Microarray Services lab using the GenomeStudio platform. Genotype data were generated using an Illumina genotyping array with approximately 730,000 SNPs. During the four years of sample collection, two versions of the array were used for genotyping. All downstream analyses used the intersection of SNPs present on both versions of the chip. We included chip version as a covariate in all association mapping models.
3
Genetics of Hispanic IBD Patients
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Data were collected with the approval of the Institutional Review Boards at the University of Miami Miller School of Medicine and Jackson Memorial Hospital (Miami, FL, USA). Information was collected prospectively from adult Gastroenterology Clinics. Patients provided detailed demographic and medical information. Foreign-born patients were asked year of immigration US immigration. Blood samples were collected and stored within the Biorepository facility at the John P. Hussman Institute for Human Genomics (HIHG) for genetic analyses. Self-identified white Hispanics and NHWs were included. IBD patient samples were genotyped on the ImmunoChip array (a custom designed Illumina iSelect panel).19 (link), 20 (link) Control samples were self-identified Hispanics who were participating in a large genetic study of cardiovascular disease at our institution. General health questions are asked of controls in order to identify and exclude any moderate to severe cases of ulcerative colitis (UC) or Crohn's disease (CD) from this sample set. These unrelated controls were also genotyped within the HIHG using a custom Illumina genotyping array.
4
Validating HLA-EMMA for Lymphocyte Immunotherapy
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To validate HLA‐EMMA, we used a previously described lymphocyte immunotherapy study cohort (n = 191).17 Briefly, this cohort consists of women that received their first lymphocyte immunotherapy from their male partner in 2009 and 2010. The HLA type of the women and their partner was determined by genotyping array (Illumina, San Diego, CA) and HLA imputation. In addition, reverse PCR sequence‐specific oligonucleotide was used to type HLA‐A and HLA‐B that were used as quality control. Antibodies against donor antigen were identified by testing sera, obtained 5 weeks (median 33 days, SD 4.5) following lymphocyte immunotherapy, with luminex single antigen bead (SAB) assays (One Lambda, Canoga Park, CA) and DSA were defined as MFI of ≥2000. HLA mismatches of which HLA second field typing could not be determined, towards which DSA were present before treatment, or that were not present in luminex SAB assay were excluded from analysis.
5
Examining SNCA Expression and Genotypes
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The SNCA gene expression dataset and analysis methods used here have been described previously49 (link). In brief, SNCA expression levels were assayed from 165 samples using quantitative real-time polymerase chain reaction. The Relative Standard Curve Method was used to transform the Ct values into quantity units. The base 10 logarithm of the SNCA expression values was used for all analyses, to ensure the normal distribution of data required by the statistical tests performed. SNP genotyping was performed as part of the US PD-GWAS Consortium meta-analysis replication sample50 (link). As described in the consortium study, the samples were genotyped at the Center for Inherited Disease Research (CIDR) using a custom Illumina genotyping array of 768 SNPs. Because the tested SNP dataset did not include the top reported PD-associated SNP rs356182 in the SNCA locus, we included rs356229 as proxy for this SNP (R2 = 0.62) in our analysis. After QC, genotyping and expression data was available for 86 cases and 41 controls for eQTL analysis. Expression models were analyzed including adjustment for disease status, sex, pH, age at death, as well as for the interaction between PMI and disease status and significance was assessed using a one sided test based on the a priori hypothesis of an association of the G-allele at rs356168 with increased SNCA expression.
6
Genotyping of Multiple Sclerosis Samples
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DNA samples were genotyped using a custom Illumina genotyping array (inclusive of the ExomeChip content) via the Illumina Infinium platform. This array targeted >300,000 genetic markers and was designed by the International Multiple Sclerosis Genetics Consortium (IMSGC) to include both ancestry informative markers24 and other single‐nucleotide polymorphisms (SNPs) specific to consortium member interests. Genotyping was conducted at the John P. Hussman Institute for Human Genomics at UM, School of Medicine.
7
Assessing MUC5B Promoter Variant
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