Molecular Analysis of Angelman Syndrome

There are different strategies for the molecular analysis of Angelman syndrome (AS), methylation analysis, FISH analysis, or linkage analysis. Most cost-effective and most easily performed, a direct test of the affected individual can be undertaken by methylation analysis at the locus D15S63. This assay distinguishes maternal and paternal loci using the methylation-sensitive restriction endonuclease, Hpa II, due to methylation of the maternal locus. The assay will detect about 80% of AS affected individuals regardless of molecular basis but does not usually distinguish the mechanism (possible mechanisms include maternal deletion, paternal uniparental disomy (UPD), or other imprinting mutation. This test involves a Southern blot analysis.

An example of an AS Southern blot analysis is shown below. A photograph of the ethidium bromide stained gel is shown in the left panel while the Southern blot of this gel is shown in the right panel. In this situation (see right panel), a deletion of maternal origin is detected in the affected individual. This is seen as a reduced intensity in the 6 Kbp Hind III maternal and paternal co-migrating fragments (Lane 3) and as a complete absence of the 6 Kbp Hind III/Hpa II fragment which is of maternal origin (Lane 6).

Left and right panels: Lane 1, DNA size markers; lane 2/5, normal control DNA sample; lane 3/6, AS patient; lane 4/7, query AS patient.

Fluorescent in situ hybridization (FISH) analysis will detect deletions ( 75% of cases) but will not detect less common uniparental disomies ( 5% of cases). An evaluation of markers in the AS critical region of parents and proband will often reveal non-transmission of alleles. Currently, CompGene tests three highly informative loci within the AS critical region (GABRB3, D15S11, D15S113). If informative, this test will reveal both deletions or UPD. PCR assay*.

NB: About 20% of AS is not due to either deletion or UPD and seems to be transmitted as an autosomal dominant with as high as a 50% recurrence risk.

In the example shown below, the parents were concerned about the recurrence of a child affected with AS despite a very low recurrence risk in AS shown to be due to a deletion of maternal origin or paternal uniparental disomy. In this case, the AS patient shows absence of maternal alleles at GABRB3 and D15S113. D15S11 was uninformative. In the current pregnancy, normal maternal and paternal contributions were observed at all three loci.