Computational protocol: The PLIN4 Variant rs8887 Modulates Obesity RelatedPhenotypes in Humans through Creation of a Novel miR-522 SeedSite

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Protocol publication

[…] A dominant model was applied to all SNPs classifying homozygotes for the major allele in one group, and carriers and homozygotes of the minor allele in another. Multivariate linear regression was used for association analyses in the GOLDN and FOS populations. To reduce variability that might obscure potential findings, multiple covariates were incorporated into our regression model, including age, sex, alcohol and tobacco smoking status, physical activity (GOLDN only), hormone use and diabetes, cholesterol and hypertension medications. To adjust for familial relationships among subjects in both populations, the lme kinship procedure was used in R, which allows the specification of the full correlation structure within pedigrees into the regression equation with random effects. To determine gene by diet interactions a SNP*dietary term was introduced into the regression equation. These tests were adjusted for total energy intake by adding total energy to the model, in addition to those mentioned above. A p value<.05 was considered significant. Response variables that did not maintain a normal distribution were log transformed to fit the normal distribution.Meta-analyses were performed with the software package Meta-Analysis helper (METAL) (www.sph.umich.edu/csg/abecasis/metal) which combines results from two or more individual studies. We used meta analysis to weight the effect size of each study by its sample size and combining Z statistics to determine an overall level of significance. [...] To identify common SNPs in the human PLIN4 locus, we searched the HapMap database for polymorphic alleles with a minor allele frequency ≥5%. The PLIN4 locus was defined as 5000 bp upstream of the predicted start codon, and 2000 bp downstream from the mRNA endpoint, a region spanning approximately 25.1 Kb. Thirteen SNPs were identified using these criteria.We chose seven of these SNPs for genotyping; two promoter (rs884164 and rs1609717), one exonic missense (rs7250947), one 3′UTR (rs8887) and three intronic (rs8102428, rs892158, and rs11673616). Analysis of the HapMap CEU population with the Haploview program determined rs892158 (rs7260518 and rs10406797), rs7250947 (rs8102428 and rs884164), rs11673616 (rs4991027) and rs1609717 (rs4807598) to be tagSNPs capturing variants in LD with an r2>0.8, predicting coverage over eleven of thirteen SNPs in the region .DNA was isolated from blood samples using DNA blood Midi kits (Qiagen, Hilden, Germany) according to the vendor's recommended protocol. Ready-made 5′ nucleic allelic discrimination assays were available from Applied Biosystems for PLIN4 SNPs rs8887, rs11673616, rs892158, rs8102428, and rs884164. We used the Applied Biosystems Custom Assay design web tool to generate functional assays for SNPs rs1609717, and rs7250947 (appliedbiosystems.com). We genotyped PLIN4 SNPs using the Taqman assays listed above on the ABIPrism 7900HT Sequence Detection System (Applied Biosystems). Standard laboratory practices were used to ensure accuracy of the data. [...] Nucleotide sequences for human, neandertal, chimpanzee, gorilla, orangutan, lemur, wild boar, cow, dog and mouse were downloaded from reference assemblies available at NCBI and aligned with ClustalW. The evolutionary history of PLIN4 was inferred using the Maximum Parsimony (MP) method. The bootstrap consensus tree inferred from 10000 replicates is taken to represent the evolutionary history of the taxa analyzed. Branches corresponding to partitions reproduced in less than 50% bootstrap replicates are collapsed. The percentage of replicate trees in which the associated taxa clustered together in the bootstrap test (10000 replicates) are shown next to the branches. The MP tree was obtained using the Close-Neighbor-Interchange algorithm with search level 3 , in which the initial trees were obtained with the random addition of sequences (100 replicates) . The analysis involved 10 nucleotide sequences. There were a total of 2447 positions in the final dataset. Evolutionary analyses were conducted in MEGA4. Total and population FST statistics for HapMap populations where estimated using the [email protected] webtool which implements the FST calculations described in Akey et al . […]

Pipeline specifications

Software tools METAL, Haploview, Clustal W, MEGA
Databases [email protected]
Applications Phylogenetics, GWAS
Organisms Homo sapiens