Wolcott-Rallison syndrome: pathogenic insights into neonatal diabetes from new mutation and expression studies of EIF2AK3

Abstract

Primers were designed to amplify all EIF2AK3 exons and splice site sequences from genomic DNA (table 1). Sequences were amplified by 35 cycles of polymerase chain reaction (PCR) using a proof-reading DNA polymerase in 50 μl reactions and purified (Qiaquick, Qiagen, Crawley, Sussex, UK). Products were sequenced using the BigDye terminator cycle sequencing kit according to the manufacturer’s instructions (Perkin-Elmer, Foster City, California, USA) and an ABI 377 sequencer (Applied Biosystems, city, county, UK). Sequences were compared to the published EIF2AK3 gene by BLAST analysis (http://www.ncbi.nlm.nih.gov/BLAST/). For potential mutations, the PCR and sequencing was repeated to confirm the result. Restriction digest analysis was also used to confirm the mutation in case 2, where the G→A substitution destroyed an HphI site.