The number of independent SNPs was determined based on the variance inflation factor (VIF) with a recursive calculation within a sliding window of 50 SNPs and pairwise r2 of 0.2. These analyses were performed selleck screening library using PLINK. Analyses of related phenotypes For each replicating SNP, we obtained association results for urinary albumin-to-creatinine ratio and microalbuminuria from our previous genome-wide association analysis [20], and for blood pressure and myocardial infarction from genome-wide association analysis from the ICBP [21] and CARDIoGRAM [22] consortia, respectively.

eSNP analysis Significant renal SNPs were searched against a database of expression SNPs (eSNP) including the following tissues: fresh lymphocytes [36], fresh leukocytes [37], leukocyte samples in individuals with Celiac disease [38], lymphoblastoid cell lines (LCL) derived from asthmatic children [39], HapMap LCL from 3 populations [40], a separate study on HapMap CEU LCL [41], peripheral blood monocytes [42], [43], adipose [44], [45] and blood samples [44], 2 studies on brain cortex [42], [46], 3 large studies of brain regions including prefrontal cortex, visual cortex and cerebellum (Emilsson, personal communication), liver [45], [47], osteoblasts [48], skin [49] and additional fibroblast, T cell and LCL samples [50]. The collected eSNP results met criteria for statistical significance for association with gene transcript levels as described in the original papers. A second expression analysis of 81 biopsies from normal kidney cortex samples was performed as described previously [51], [52].

Genotyping was performed using Affymetrix 6.0 Genome-wide chip and called with GTC Software (Affymetrix). For eQTL analyses, expression probes (Affymetrix U133set) were linked to SNP probes with >90% call-rate using RefSeq annotation (Affymetrix build a30). P values for eQTLs were calculated using linear multivariable regression in both cohorts and then combined using Fisher’s combined probability test (see also [52]). Pairwise LD was calculated using SNAP [53] on the CEU HapMap release 22. Zebrafish functional experiments Zebrafish were maintained according to established IACUC protocols. Briefly, we injected zebrafish embryos with newly designed (mpped2, ddx1) or previously validated (casp9 [54]) morpholino antisense oligonucleotides (MO, GeneTools, Philomath OR) at the one-cell stage at various doses.

We fixed embryos in 4% PFA at the appropriate stages for in situ hybridization (http://zfin.org/ZFIN/Methods/ThisseProtocol.html). Different anatomic regions of the kidney were visualized using a panel of 4 established markers: pax2a (global kidney marker) [15], nephrin (podocyte marker) [16], slc20a1a (proximal Drug_discovery tubule) [17], and slc12a3 (distal tubule marker) [17]. Abnormalities in gene expression were independently scored by two investigators.