Pyrosequencing technology in Clinical Genetics Applications

Pyrosequencing technology produces valuable data in a range of studies of clinical genetic syndromes. Here, we illustrate the performance of Pyrosequencing technology in quantitative measurement of methylation in Prader-Willi & Angelmann syndromes, the identification of multiple polymorphisms in the APOE gene, simple genotyping of adult hypolactasia, and a single test to characterise the complex cancer mutations involved in Multiple Endocrine Neoplasia (MEN2).

Prader-Willi / Angelmann are developmental syndromes usually caused by deletion of a differentially methylated DNA region on the human chromosome 15q11-13. Different patterns of DNA methylation are detected depending on which chromosome is deleted. A maternally deleted chromosome leads to hypomethylation, whereas hypermethylation is seen when the paternal chromosome is deleted.

Pyrosequencing technology is a simple and fast technique to detect the two syndromes, providing accurate assessment of methylation 30 minutes after PCR.

Illustration of PyroMark Prader-Willi/Angelman Syndromes assay results. Blue columns indicate
quantified CpG sites. Yellow columns indicate QC for completion of the bisulfite treatment (a C followed
by an A in the original sequence). Pyrogram trace shows the reverse assay of the top strand.

APOE - Multiple SNPs in one assay

Illustration of PyroMark APOE assay results. Blue columns indicate the analyzed SNPs.

ApolipoproteinE (apoE) is a plasma protein involved in lipid transport and lipoprotein metabolism. The APOE gene is polymorphic with three common alleles designated epsilon-2, epsilon-3 and epsilon-4. These genetic variations result in amino acid substitutions at positions 112 and 158 of the 299 amino acid protein. The proportion of the three alleles differs between populations with the epsilon3 allele being the most prevalent form.

Pyrosequencing technology provides a fast and simple technique to clearly and reproducible distinguish between the three different alleles in one single assay.