Mutation AnalysisWe designed primer

Mutation AnalysisWe designed primers with the Primer 3 program to amplify the full region spanned from the 50 UTR region to exon 3. This span starts from 1.5 kb upstream of exon 1a to 62 bp downstream from exon3. We also amplified all coding regions from exon 4 to exon 9, including splice sites, and the selected regions from intron 3 and 30 UTR. For PCR reactions, the AmpliTaq Gold DNA polymerase(Applied Biosystems, Foster City, CA) or Fast Taq polymerase(Roche,Indianapolis, IN) was used according to manufacturer’s instructions. Both patient and healthy-control genomic-DNA samples served as templates for PCR amplifications. Each PCR product of probands and controls was screened by heteroduplex analysis with the use of denaturing high-performance liquid chromatography (DHPLC WAVE, Transgenomic)26,27 or directly sequenced with the use of ABI 3700 capillary automated-sequencing system (Applied Biosystems, Foster City, CA).Suspected variants were subjected to PCR at least twice and confirmed by digestionor sequences of parents.