Figure 5.

Molecular diagnostic flowchart. Methylation analysis is performed by combined bisulfite restriction analysis, bisulfite sequencing, multiple ligation probe amplification (MLPA) using SALSA MLPA kit ME032 UPD7/UPD14 (MRC-Holland, Amsterdam, The Netherlands) or pyrosequencing. Parent-of-origin analysis is carried out by microsatellite analysis or single-nucleotide polymorphism (SNP) array. Deletion analysis is performed by MLPA, fluorescence in situ hybridization or array comparative genomic hybridization. Methylation and deletion analyses need DNA samples of patients alone, whereas parent-of-origin analysis requires DNA samples of patients and their parents. MLPA is utilized for deletion analysis and methylation analysis before and after digestion of the genomic DNA samples with a methylation-sensitive restriction enzyme HhaI, respectively.