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Rapid detection of FGFR mutations in syndromic craniosynostosis by temporal temperature gradient gel electrophoresis.

Several autosomal dominant craniosynostosis syndromes, including Crouzon, Pfeiffer, Apert, and Jackson-Weiss syndromes, are caused by mutations in the fibroblast growth factor receptor (FGFR) gene family (1). Mutations are not uniformly dispersed among these genes, and most mutations described to date are in exons IIIa and 111c of the FGFR2 gene (2, 3). The advent of molecular analysis of these genes has allowed confirmatory testing in ambiguous cases and for those families desiring prenatal diagnosis. However, molecular analysis is difficult at present, especially for Crouzon syndrome, where current methods rely on the sequencing of the aforementioned exons. We report the rapid and inexpensive detection of FGFR mutations by a relatively novel screening assay, temporal temperature gradient gel electrophoresis (TTGE).

TTGE is a heteroduplex detection method similar to denaturing gradient gel electrophoresis (DGGE). Like DGGE, but often unlike single-strand conformation polymorphism and heteroduplex analysis using mutation detection enhancement gels, TTGE is highly sensitive (4-6). However, TTGE is simpler than DGGE in avoiding the use of a chemical denaturing gradient gel and GC clamps (4). In TTGE, a homogeneous gel is bathed in a tank of buffer in which the temperature of the entire unit increases linearly throughout the electrophoretic run (5). After PCR, the products are heated and allowed to cool gradually, which leads to the formation of heteroduplex DNA if sequence heterogeneity is present. In dominant diseases, such as FGFR mutations that cause craniosynostosis syndromes, the sequence difference between the two alleles in the heteroduplex causes a physical bulge at the site of the sequence mismatch, which produces a localized lowering of the melting temperature. As the temperature of the gel increases during TTGE, the specific temperature is reached at which the area around the mismatch melts in the heteroduplex DNA, which in turn reduces the mobility of the heteroduplex and separates it from the homoduplex DNA. In the absence of any mutation, a single distinct band is seen on the gel (wild-type homoduplex DNA only; Fig. 1). However, in the presence of a mutation, up to four bands can be visualized: the wild-type homoduplex, the mutant homoduplex, and the two heteroduplexes. Frequently, the two homoduplexes (and rarely, the two heteroduplexes) are not separated, producing a total of three (or two) bands (Fig. 1). In de novo mutations, the additional bands visualized in the patient are absent in both parents.

A total of eight patients with a clinical diagnosis of one of the FGFR-related craniosynostosis syndromes were studied (Table 1). Total DNA was isolated from the peripheral blood of each patient and, when available, both parents, using a Puregene DNA Isolation Kit (Gentra Systems). PCR amplification was performed with a GeneAmp PCR System 9700 (Applied Biosystems) in a final volume of 40 [micro]L with the following primer pairs: FGFR2 exon IIIa, 5'-TGACA000TCTGACAACACAAC-3' (forward) and 5'-GGAAATCAAAGAACCTGTGGC-3' (reverse) (7); and FGFR2 exon IIIc, 5'-CACAATCATTCCTGTGTCGT-3' (forward) and 5'AACCCAGAGAGAAGAACAGTA-3' (reverse) (8). The reaction mixture consisted of 1 [micro]L of DNA (-250 ng), 0.2 [micro]L of Taq DNA polymerase (5 U/[micro]L; Promega), 4 [micro]L of 10X PCR buffer (Promega), 2 [micro]L of 25 mmol/L Mg[Cl.sub.2] (Promega),1 [micro]L of 8 mmol/L dNTP (Promega), and 1 [micro]L of 20 mmol/L primers. PCR-grade water was added to a final volume of 40 [micro]L. Amplification was performed after a denaturation process of 4 min at 94[degrees]C, followed by 36 cycles each of 30 s at 94[degrees]C, 45 s at 58[degrees]C, and 45 s at 72[degrees]C, and a final extension period of 7 min at 72[degrees]C. Amplified PCR products were heated at 94[degrees]C for 4 min and allowed to cool gradually at room temperature to create heteroduplexes. The samples were then loaded onto a 7% polyacrylamide gel (Bio-Rad Laboratories) with 6 mol/L urea in 1.25X Tris-acetate-EDTA buffer, and TTGE was performed with a D-Code Universal Mutation Detection System (Bio-Rad) (5). TTGE conditions for each segment were determined by computer simulation using the MacMelt program (Bio-Rad) and adjusted by experimentation. Initial, final, and ramp rate temperatures for TTGE were as follows: for IIIa, 58[degrees]C, 65[degrees]C, and 2.4[degrees]C/h; and for IIIc, 56[degrees]C, 64[degrees]C, and 2.4[degrees]C/h. Ethidium bromide-stained TTGE gels were visualized under ultraviolet light and imaged with a High Performance CCD digital camera (Cohu). For quality assurance, every gel was run concurrently with at least one positive control. Positive results were repeated from the original DNA sample to rule out the possibility of a Taq-induced error. Sequencing was performed with a dye terminator cyclosequencing kit (PE Applied Biosystems) and an ABI 373A DNA Sequencer (Applied Biosystems).

In six of the eight children, heteroduplex bands were clearly visible by TTGE in one exon each (Fig. 1), including three of four Crouzon, three of three Apert, and zero of one Pfeiffer cases. In the three of the six positive cases in which parental blood samples were available, single bands were present in both parents, indicating the presence of a de novo mutation in the child (Fig. 1). Both exons were sequenced in all eight patients, and in all six cases identified as positive by TTGE, sequencing identified a mutation that was reported previously in at least one patient with a craniosynostosis syndrome (Table 1). No mutation was found when both exons in the two cases identified as negative by TTGE were sequenced.


Our preliminary data demonstrate that TTGE is an accurate, rapid, and inexpensive method to detect FGFR mutations in patients with selective craniosynostosis syndromes. The sensitivity on testing only these two exons is 75%, a value that will likely increase as we expand the region of screening. For example, many individuals with Pfeiffer syndrome have a common mutation in FGFR1 (1), which likely explains our failure to find a mutation in our one Pfeiffer case. As many as 45 patients can be screened by TTGE per exon by a single technician and electrophoresis apparatus in 1 day. Only those patients in which heteroduplex bands are found require sequencing and only in the exon identified. The screening of parental samples to rule out the presence of a polymorphism may be appropriate in the case of a previously unreported mutation. The unlikely scenario that both parents are homozygous for different polymorphisms can be excluded by performing TTGE on a parental DNA mixture. We believe that TTGE is suitable for the screening of patients at risk for FGFR mutations in a clinical setting.

TTGE is a sensitive and inexpensive screening assay for heteroduplex DNA that likely can be applied for mutation detection with many different genes. We have been using this technique primarily for screening mitochondrial DNA and studied the FGFR genes as a model for the use of TTGE in autosomal dominant disease. The use of TTGE to screen for COL2A1 mutations in Kniest dysplasia, another autosomal dominant disorder, was presented recently as an abstract (9). Disorders in which application of this method are likely to be most suitable include those caused by a large number of different mutations that are largely confined to relatively small areas of the gene.

This work was supported by a grant from the Childrens Hospital Los Angeles Research Institute.


(1.) Wilkie AOM. Craniosynostosis: genes and mechanisms. Hum Mol Genet 1997;6:1647-56.

(2.) Oldridge M, Wilkie AOM, Slaney SF, Poole MD, Pulleyn U, Rutland P, et al. Mutations in the third immunoglobulin domain of the fibroblast growth factor receptor-2 gene in Crouzon syndrome. Hum Mol Genet 1995;4:1077-82.

(3.) Oldridge M, Lunt PW, Zackai EH, McDonald-McGinn DM, Muenke M, Moloney DM, et al. Genotype-phenotype correlation for nucleotide substitutions in the IgII-IgIII linker of FGFR2. Hum Mol Genet 1997;6:137-43.

(4.) Cotton RGH. Slowly but surely towards better scanning for mutations. Trends Genet 1997; 13:43-6.

(5.) Zoller P, Redilla-Flores T. Temporal temperature gradient gel electrophoresis of cystic fibrosis samples on the Dcode system. US/EG Bulletin 2103. Hercules, CA: Bio-Rad Laboratories, 1996.

(6.) Chen T-J, Boles RG, Wong L-J. Detection of mitochondrial DNA mutations by temporal temperature gradient gel electrophoresis. Clin Chem 1999;45: 1162-7.

(7.) Park W-J, Meyers GA, Li X, Theda C, Day D, Orlow SJ, et al. Novel FGFR2 mutations in Crouzon and Jackson-Weiss syndromes show allelic heterogeneity and phenotypic variability. Hum Mol Genet 1995;4:1229-33.

(8.) Jabs EW, Li X, Scott AF, Meyers G, Chen W, Eccles M, et al. Jackson-Weiss and Crouzon syndromes are allelic with mutations in fibroblast growth factor receptor 2. Nature 1994;8:275-9.

(9.) Lin T, Wilkin DJ, Chen T, Francomano CA, Wong LC. Identification of mutations and polymorphisms in the COL2A1 gene by temporal temperature gradient gel electrophoresis. Am J Hum Genet 1998;63:A371.

Tomoyasu Higashimoto, [1] Shoji Yano, [1,3] Masamichi Ito, [4,6] Noreen C. Clark-Sheehan [2,5] Linda Cowan, [1] and Richard G. Boles [1,3] *

(Divisions of [1] Medical Genetics and [2] Plastic Surgery, Childrens Hospital Los Angeles, Los Angeles, CA 90027; Departments of [3] Pediatrics, [4] Obstetrics and Gynecology, and [5] Surgery, and [6] Institute for Genetic Medicine, University of Southern California School of Medicine, Los Angeles, CA 90033; * address correspondence to this author at: Medical Genetics, Box 90, Childrens Hospital Los Angeles, 4650 Sunset Blvd., Los Angeles,CA90027;fax323-665-5937,e-mailrboles@chla.
Table 1. FGFR2 mutations identified by TTGE.

Mutation Our patient's phenotype Reported phenotypes

Ser252Trp (C767G) A (b) A
Ser252Trp (C767G) A A
Cys342Tyr (G1037A) C C, P
Cys342Ser (G1037C) C C, P
Ser347Cys (C1052G) C C
Ser351Cys (C1064G) A U

(a) Wilkie (1).

(b) A, Apert syndrome; C, Crouzon syndrome; P, Pfeiffer syndrome; U,
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Title Annotation:Technical Briefs
Author:Higashimoto, Tomoyasu; Yano, Shoji; Ito, Masamichi; Clark-Sheehan, Noreen C.; Cowan, Linda; Boles, R
Publication:Clinical Chemistry
Date:Nov 1, 1999
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