Such studies have also led to the classification of novel syndromes diagnosed at the molecular level, such as Muenke syndrome, which is related to the FGFR3 P250R mutation (3,4).
To optimize assay conditions, we used 20 DNA control samples carrying the following 10 mutations: S252W (755C>G), P253R (758C>G), F276V (826T>G), C278F (833G>T), C342R (1024T>C), C342Y (1025G>A), C342S (1025G>C), A344P (1030G>C), and S347C (1040C>G) mutations in the FGFR2 gene and the P250R (749C>G) mutation in the FGFR3 gene.
Seven of the sequence variants were located either at the 5' terminus (P250R, S252W, F276V, C342R, S347C) or at the 3' terminus (A344P, P253R) of the probe, because a base-stacking interaction with an adjacent stabilizer oligonucleotide permitted a more stable probe-to-template interaction in this format.
FGFR3 exon 7 (where the P250R mutation is located) could not be included in the multiplexing PCR format because of a high sequence similarity with FGFR2 exon IIIa (location of the P253R mutation).
Notably, the craniosynostosis associated with the P250R FGFR3 mutant has previously been referred to as Muenke syndrome (4, 18, 19).
The FGFR3 P250R mutation and TWIST1 mutations have been identified in 50% of Saethre-Chotzen cases and in 27.7% of isolated plagiocephaly cases and 31.8% of brachicephaly cases.
An interesting problem we faced in the present work was analyzing mutations in highly homologous DNA regions, such as in FGFR3 exon 7 and FGFR2 exon IIIa, where the P250R and P253R variants are located, respectively.
To overcome this problem, it was sufficient first to address and probe the critical amplicon (FGFR3 exon 7) with the wild-type and mutant reporter set for the P250R mutation and subsequently to address the duplex PCR of FGFR2 exons IIIa and IIIc to allow serial probe hybridization with all probe sets on the same pad.
FGFR3 P250R mutation increases the risk of reoperation in apparent 'nonsyndromic' coronal craniosynostosis.