Prader-Willi syndrome (PWS) is a complex genetic disorder that includes short stature, mental retardation or learning disabilities, incomplete sexual development, characteristic behaviour problems, low muscle tone, and an involuntary urge to eat constantly, which, coupled with a reduced need for calories, leads to obesity.

About 1 in 14,000 people in the U.S. are estimated to have PWS, and the birth rate may be even higher. Prader-Willi syndrome is one of the 10 most common conditions seen in genetics clinics.

PWS is caused by loss of expression of genes located on segment q11-13 on the paternally derived chromosome 15. Approximately 70% of patients with PWS have a deletion involving bands 15q11.2-q13.

When the expression from the same segment is lost from the maternally derived chromosome 15, the Angelman syndrome (AS) arises.

This methylation analysis assay detects deletion of segment q11-13 on chromosome 15.

Principles of the procedure

The DNA has to be bisulfite-treated before PCR amplification. Kits for the procedure are commercially available, e.g. EZ DNA Methylation Kit™ from Zymo Research. During bisulfite treatment of the genomic DNA, unmethylated Cytosine is converted to Uracil, whereas methylated Cytosine remains unchanged. Using PCR, Uracil is amplified to Thymine, whereas methylated Cytosine is amplified as Cytosine. Discrimination between methylated Cytosine and unmethylated Cytosine is achived by analyzing the CpG sites as C/T SNP positions.>

A single PCR fragment spanning a region frequently deleted in PWS and AS is amplified and the degree of methylation of four CpG sites is analyzed in a single Pyrosequencing reaction. A control for completion of bisulfite treatment is included in the assay. A Cytosine followed by an Adenine in the normal sequence should be quantified as 100%T if fully converted during bisulfite treatment.

See figure 1 for an illustration of the assay.

Illustration of the Prader-Willi/Angelman assay. PCR primers are shown as solid arrows and the sequencing primer as a dashed arrow. RP: reverse primer; FPB: biotinylated reverse primer; Seq: sequencing primer; X: CpG sites. Q indicates a control for completion of bisulfite treatment.

If the maternal chromosome region is deleted, methylation is close to 0% and if the paternal chromosome region is deleted, methylation is close to 100%.

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