Novel SNP at the common primer site of exon IIIa of FGFR2 gene causes error in molecular diagnosis of craniosynostosis syndrome

Most mutations in Crouzon, Pfeiffer, and Apert syndromes are in the extrace llular, third immunoglobulin-like domain and adjacent linker regions (exons IIIa and IIIc) of the fibroblast growth factor receptor 2 (FGFR2) gene. Us ing the published primers for PCR, a patient with Crouzon syndrome was foun d to be homozygous for a mutation that results in a Q289P amino acid substi tution in FGFR2. Two additional patients; one with Apert syndrome and P253R mutation, the other with Pfeiffer syndrome and S267P mutation, also appear ed to be homozygous. Using a new primer located 146 bp 5' of exon IIIa for PCR followed by sequencing revealed an A to G polymorphism at -61 position of exon IIIa. All three patients were heterozygous for both the mutation an d the polymorphism. These results indicate that the polymorphism and the mu tation are not on the same chromosome. The single nucleotide polymorphism i s located at the second to the last base of the Tend of the published prime r. This primer mismatch caused the failure of amplification of the normal c hromosome and thus, the apparent homozygosity. The frequency of this novel polymorphism was determined to be 0.03 by studying 326 chromosomes from the general population. We propose that a new primer should be used for mutati onal analysis of exon IIIa of FGFR2 to avoid misdiagnosis caused by primer mismatch. (C) 2001 Wiley-Liss, Inc.