Leiden DNA Methylation Analysis

Background: The following procedure describes the detailed DNA methylation analysis process of liquid DNA samples by methylation-sensitive Southern blot performed at the Leiden University Medical Center. Detailed DNA methylation studies showed D4Z4 hypomethylation at the BsaAI and FseI restriction sites in the proximal D4Z4 repeat unit in patients with FSHD; both in FSHD patients with a D4Z4 contraction (4q-linked FSHD or FSHD1) and in a small group of FSHD patients without a D4Z4 contraction on the pathogenic haplotype (phenotypic FSHD or FSHD2).
Liquid DNA samples should be of high quality, especially for complete FseI digestion. This analysis only measures the D4Z4 methylation level on all chromosome 4 alleles, because of inclusion of the restriction enzyme BlnI to all digestion reactions. Upon full digestion, appropriate ratios of signal intensities should be obtained; e.g. in case of a disomic individual with two 4-type D4Z4 repeats and two 10-type D4Z4 repeats the ratio of signal intensities between all bands representing chromosome 4q and all bands representing chromosome 10q should be 1:1.

Relevant References:

1. Overveld PG, Lemmers RJ, Sandkuijl LA, Enthoven L, Winokur ST, Bakels F, Padberg GW, van Ommen GJ, Frants RR, van der Maarel SM. Hypomethylation of D4Z4 in 4q-linked and non-4q-linked facioscapulohumeral muscular dystrophy. Nat Genet. 2003 Dec;35(4):315-7.

 

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