DNA was isolated from whole blood in all patients. In six, we were able to obtain additional samples, including saliva, buccal epithelial cells, urine, fresh blood for isolation of circulating lymphocytes, and myeloid cells. Isolation of neutrophils, monocytes, and T and B lymphocytes was performed with magnetic nanoparticles (Stemcell Technologies, Inc., Manchester, UK) following the manufacturer’s protocol. DNA was extracted from isolated cells using Qiagen Investigator DNA extraction Kit (Qiagen Ltd., Manchester, UK).
NLRP3/CIAS1 (exons 3, 4, and 6) [NCBI RefSeqGene NC_000001.10 (LRG_197)] and NLRP12 (exon 3) [NCBI RefSeqGene NC_000019.9] genes were amplified by PCR and analyzed by Sanger sequencing using the protocol described previously (16 (link)). The chromatograms were analyzed on the ABI 3130xl Genetic Analyser using Sequencing Analysis Software version 5.4.
Amplicon-based deep sequencing was performed to amplify all exons of the NLRP3 gene using specific pair primers as previously described (17 (link)). All PCR amplicons were deep sequenced (mean coverage 3,500×) on different platforms (GS Junior 454, PGM IonTorrent and Illumina MiSeq). The sequences were analyzed using the Amplicon Variant Analyzer software (Roche, Switzerland).
NLRP3/CIAS1 (exons 3, 4, and 6) [NCBI RefSeqGene NC_000001.10 (LRG_197)] and NLRP12 (exon 3) [NCBI RefSeqGene NC_000019.9] genes were amplified by PCR and analyzed by Sanger sequencing using the protocol described previously (16 (link)). The chromatograms were analyzed on the ABI 3130xl Genetic Analyser using Sequencing Analysis Software version 5.4.
Amplicon-based deep sequencing was performed to amplify all exons of the NLRP3 gene using specific pair primers as previously described (17 (link)). All PCR amplicons were deep sequenced (mean coverage 3,500×) on different platforms (GS Junior 454, PGM IonTorrent and Illumina MiSeq). The sequences were analyzed using the Amplicon Variant Analyzer software (Roche, Switzerland).
Free full text: Click here
