Mutation Cloning
A 1482 bp region covering the mutation and an informative heterozygous (C/G) SNP, rs2078304, in patient M4 was amplified by LR-PCR and cloned with the Copy Control Cloning Kit (Epicenter Biotechnologies, Madison, WI, USA). Clone DNA was isolated with the Qiprep Miniprep Kit (QIAGEN, Valencia, CA, USA) and sequenced by conventional Sanger di-deoxynucleotide sequencing (Lone Star Labs, Houston, TX, USA).