Congenital Central Hypoventilation Syndrome / Ondine syndrome

1) To detect non-polyalanine repeat expansion mutations (NPARMs), and polyalanine repeat expansion mutations (PARMs), Sanger sequencing of the amplicons is performed.

Large copy number variations (deletions, insertions, duplications) cannot be excluded with this analysis.

2) PHOX2B has two polyalanine repeat regions in exon 3. Therefore the amplicons are also analysed by capillary electrophoresis on a Fragment Analyzer device. 

In most CCHS individuals with neonatal onset, polyalanine repeat expansion mutations (PARMs) of 25 or more GCN repeats can be found (the N in GCN represents any nucleotide: A/C/G/T). Also the 35-bp and 38-bp NPARM recurrent out-of-frame deletions within the GCN repeat region can be detected.

3) As PHOX2B partial and whole-gene deletions have also been reported, MLPA analysis (multiplex ligation dependent probe amplification) is performed for detection of copy number variations (CNVs) in the PHOX2B gene (MRC Holland, P318-Hirschsprung-2, version A2).