Sensitive detection of polyalanine expansions in PHOX2B by polymerase chain reaction using bisulfite-converted DNA

Congenital central hypoventilation syndrome, also known as Ondine's curse, is characterized by idiopathic abnormal control of respiration during sleep. Recent studies indicate that a polyalanine expansion of PHOX2B is relevant to the pathogenesis of this disorder. However, it is difficult to detect the repeated tract because its high GC content inhibits conventional polymerase chain reaction (PCR) amplification. Here, we describe a bisulfite treatment for DNA in which uracil is obtained by deamination of unmethylated cytosine residues. Deamination of DNA permitted direct PCR amplification that yielded a product of 123 bp for the common 20-residue repetitive tract with replacement of C with T by sequencing. It settled allele dropouts accompanied by insufficient amplification of expanded alleles. The defined procedure dramatically improved detection of expansions to 9 of 10 congenital central hypoventilation syndrome patients examined in a previous study. The chemical conversion of DNA before PCR amplification facilitates effective detection of GC-rich polyalanine tracts.