Molecular Analysis of Prader-Willi Syndrome

There are different strategies for the molecular analysis of Prader-Willi syndrome (PWS), 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 virtually all PWS affected individuals regardless of molecular basis but does not always distinguish the mechanism (possible mechanisms include paternal deletion, maternal uniparental disomy (UPD), or other imprinting mutation. This test involves a Southern blot analysis.

An example of an PWS 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), uniparental maternal disomy is detected in one of the query PWS patients. This is seen as an unaltered intensity in the 6 Kbp Hind III maternal and paternal co-migrating fragments (Lane 3) and as an unaltered intensity in the 6 Kbp maternal Hind III/Hpa II fragment (lane 6; yellow arrow) and as a complete absence of the 4.4 Kbp Hind III/Hpa II fragment which is of paternal origin (Lane 6; red arrow). The patient sample shown in lane 4/7 appeared normal.

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

Fluorescent in situ hybridization (FISH) analysis will detect deletions ( 75% of cases) but will not detect uniparental disomies ( 25% of cases). An evaluation of markers in the PWS critical region of parents and proband will often reveal non-transmission of alleles. Currently, CompGene tests three highly informative loci within the PWS critical region (GABRB3, D15S11, D15S113). If informative, this test will reveal both deletions or UPD. PCR assay*

In the example shown below, the PWS patient shows absence of paternal alleles at GABRB3 and D15S113. D15S11 was uninformative. In addition, both copies of the maternal chromosome 15 loci have been inherited by the affected child, a case of maternal uniparental heterodisomy.