Computational protocol: PHKA2 mutation spectrum in Korean patients with glycogen storage disease type IX: prevalence of deletion mutations

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Protocol publication

[…] Human genomic DNA was prepared from frozen white blood cells using a Wizard genomic DNA purification kit (Promega, Madison, WI, USA) according to the manufacturer’s recommendations. All 33 exons and the flanking regions of the PHKA2 gene were amplified by polymerase chain reaction (PCR) using primers designed by the authors (Table ) with a thermal cycler (Model 970; Applied Biosystems, Foster City, CA, USA). Direct sequencing of the DNA was performed using the ABI Prism 3100 Genetic Analyzer (Applied Biosystems) with the BigDye Terminator Cycle Sequencing-Ready Reaction Kit (Applied Biosystems). Nucleotides are numbered from the first adenine of the ATG translation initiation codon in the PHKA2 cDNA Reference Sequence NM_000292.2.To detect single or multiple exon deletions, a multiplex PCR method was performed using primers designed by the authors (Table ). All tests were performed concurrently on negative control samples from healthy individuals.Additionally, a comprehensive review of the literature on previously reported PHKA2 mutations in Asian populations was conducted. The Leiden Open Variation Database (LOVD, and Human Gene Mutation Database (HGMD, h were checked for previously reported sequence variants. Variants identified in this study were checked through public databases. Common and rare variants present in the PHKA2 gene in Korean population could be obtained from the Korean Reference Genome Database ( and compared with other ethnic populations through the Exome Aggregation Consortium (ExAC), which aggregates over 60,000 human exomes. The 1000 Genomes Project database (, the National Heart, Lung, and Blood Institute (NHLBI) Exome Sequencing Project (ESP) database (, and the NCBI database of Single Nucleotide Polymorphisms (dbSNPs) were also checked for previously reported sequence variants.The pathogenicity of missense variants was evaluated by in silico analyses using Sorting Intolerant from Tolerant (SIFT) (, and Polymorphism Phenotyping v.2 (PolyPhen-2) ( prediction programs. Human Splice Finder software ( was used to predict splicing signals []. […]

Pipeline specifications

Software tools Biobase, SIFT, PolyPhen
Applications Miscellaneous, WES analysis
Organisms Homo sapiens
Diseases Glycogen Storage Disease, Glycogen Storage Disease Type II