Germline mutation in the FGFR3 gene in a TWIST1‐negative family with saethre‐chotzen syndrome and breast cancer

In a recent study published in Genes Chromosomes and Cancer (Sahlin et al., 2007), we showed that women with the Saethre-Chotzen syndrome, caused by mutations in the basic helix-loop-helix transcription factor gene TWIST1 (El Ghouzzi et al., 1997; Howard et al., 1997), have an increased risk of breast cancer. In 15 Saethre-Chotzen families, we found that >50% of female carriers older than 25 had developed breast cancer. These findings identified breast cancer as an important and previously unrecognized symptom typical of this syndrome and indicated that TWIST1 may be a novel breast cancer susceptibility gene. Saethre-Chotzen syndrome is one of the most common craniosynostosis (premature fusion of one or more cranial sutures) syndromes characterized by coronal synostosis, S-shaped blepharoptosis, low set ears with typical apical cartilage deformity, cutaneous syndactyly of dig II-III, typical nasal deformity, and low frontolateral hairline (references in Pantke et al., 1975; Sahlin et al., 1994, 2007). It is an autosomal dominantly inherited syndrome with a variable expressivity (Pantke et al., 1975; Reardon and Winter, 1994). Genetic analyses of both familial and sporadic cases have shown that Saethre-Chotzen is genetically heterogeneous (Paznekas et al., 1998; Chun et al., 2002). The majority of cases are caused by mutations in TWIST1 but there are also welldocumented cases caused by mutations in the FGFR2 or FGFR3 genes. Approximately 80% of patients with a Saethre-Chotzen phenotype have mutations in one of these three genes (Chun et al., 2002). As part of our continued studies of SaethreChotzen syndrome and its recently established association with breast cancer, we have now identified and characterized a TWIST1-negative family with a Saethre-Chotzen phenotype and breast cancer (Family 7 in Sahlin et al., 2007). A 61-year-old woman (designated mother), whose father also had Saethre-Chotzen syndrome, presented with typical clinical signs of Saethre-Chotzen, including unicoronal synostosis, symmetrical cutaneous syndactylies of the hands and feet (dig II-III), low set ears with typical apical cartilage deformity, and hearing impairment. At the age of 55, she developed breast cancer. Six years after surgery, she is alive and well. Nucleotide sequence analysis of DNA isolated from a blood sample revealed no mutation of the TWIST1 gene (data not shown). As Saethre-Chotzen syndrome may also be caused by mutations in the FGFR2 and FGFR3 genes (see earlier), we also screened these genes for possible mutations. All exons of the FGFR2 (19 exons) and FGFR3 (19 exons) genes were sequenced using bidirectional DNA sequencing essentially as described earlier (Persson et al., 2008; PCR primers for DNA sequencing are available on request). Briefly, sequence PCR was performed using the BigDye Terminator v3.1 Cycle Sequence Kit (Applied Biosystems, Foster City, CA). The sequencing products were separated with gel electrophoresis on a 3730 DNA analyser (Applied Biosystems) and the output data were analyzed using SeqScape v 2.5 (Applied Biosystems). Analysis of the resulting sequences revealed a mutation in FGFR3 exon 7, i.e., 749C>G (Pro250Arg; P250R), in the DNA of the mother (Fig. 1). This missense mutation was present in a heterozygous state. Furthermore, multiplex ligation-dependent probe amplification (MLPA) analysis was performed using the Salsa MLPA Kit P080 (MRC-Holland, The Netherlands), as described by the manufacturer. This kit is developed for detection of large deletions in a set of genes known to be involved in craniofacial disorders (ALX4, TWIST1, RUNX2, MSX2, FGFR1/2/3/4, and EFNB1). A probe for FGFR3 exon 7 gave a signal of half the intensity for the DNA of the mother as compared to healthy control DNA (Fig. 1). It is described by the manufacturer of the MLPA kit (MRC-Holland) that