Puregene system

This cross-sectional case-control study was designed to examine the genetics of T2D and ESKD in African Americans [5] (link). Patients with T2D-ESKD were recruited from dialysis facilities. T2D was diagnosed in African Americans who reported developing T2D after the age of 25 years and did not receive only insulin therapy since diagnosis. In addition, cases had at least one of the following three criteria for inclusion: a) T2D diagnosed at least 5 years before initiating renal replacement therapy, b) background or greater diabetic retinopathy by self-report and/or c) ≥100 mg/dl proteinuria on urinalysis in the absence of other causes of nephropathy. Unrelated African American controls without a current diagnosis of diabetes or renal disease were recruited from the community and internal medicine clinics. All T2D-ESKD cases and controls lacking T2D and ESKD were born in North Carolina, South Carolina, Georgia, Tennessee or Virginia. DNA extraction was performed using the PureGene system (Gentra Systems; Minneapolis, MN).

DNA in AA-DHS participants was extracted from peripheral blood using the PureGene system (Gentra Systems, Minneapolis, MN). The AA-DHS GWAS utilized the Illumina 5 M chip; the JHS GWAS genotyping was performed with the Affymetrix 6.0 chip. Genotypic data in both studies were imputed to 1000 Genomes (phase 1 version 3, cosmopolitan panel) [21 (link)].

DNA from AA-DHS participants was extracted from peripheral blood using the PureGene system (Gentra Systems, Minneapolis, MN). The AA-DHS GWAS utilized the Illumina 5M chip. Quality control (QC) checks in AA-DHS led to the exclusion of 12 individuals from the analyses: six had call rates <95%, two had discordant self-reported and genetically determined sex, one had a heterozygosity score outside of the mean ±4 times the standard error interval, two had the same sample identifiers and one had 100% European ancestry. GWAS analyses were performed on up to 697 individuals.
Imputation was performed using IMPUTE2 with phased haplotype data obtained from SHAPEIT2 [16 (link)]. Directly genotyped variants with a Hardy-Weinberg Equilibrium (HWE) p-value ≥1x10^-04 and a call rate ≥95% were included and imputation was based on 3,436,913 autosomal variants. The multi-ethnic 1,000 Genomes Phase I integrated variant set release (v3) was used as the reference panel. Statistical analysis was performed for imputed variants that had an info score above 50%, a minor allele frequency (MAF) >1% and a HWE p-value ≥1x10^-04.

Blood samples were collected by venipuncture at the antecubital fossa during examination 4 (1991–1993), examination 5 (1994–1996) or examination 6 (1997–1999) and stored at − 70 °C. Frozen buffy coat samples were used for extraction of DNA. The PureGene system (Gentra Systems, Inc.) was used to isolate total cellular DNA, which was quantified using PicoGreen staining (Molecular Probes).
Procedures performed were in compliance with institutional guidelines and were approved by the Kuakini Medical Center Institutional Review Board. Written informed consent was obtained at all examinations from study participants or from family representatives if participants were unable to provide consent.