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. 2000 Mar;66(3):768-77.
doi: 10.1086/302831.

Paternal origin of FGFR2 mutations in sporadic cases of Crouzon syndrome and Pfeiffer syndrome

R L Glaser  1 , W Jiang, S A Boyadjiev, A K Tran, A A Zachary, L Van Maldergem, D Johnson, S Walsh, M Oldridge, S A Wall, A O Wilkie, E W Jabs
Affiliations

Paternal origin of FGFR2 mutations in sporadic cases of Crouzon syndrome and Pfeiffer syndrome

R L Glaser et al. Am J Hum Genet. 2000 Mar.

Abstract

Crouzon syndrome and Pfeiffer syndrome are both autosomal dominant craniosynostotic disorders that can be caused by mutations in the fibroblast growth factor receptor 2 (FGFR2) gene. To determine the parental origin of these FGFR2 mutations, the amplification refractory mutation system (ARMS) was used. ARMS PCR primers were developed to recognize polymorphisms that could distinguish maternal and paternal alleles. A total of 4,374 bases between introns IIIa and 11 of the FGFR2 gene were sequenced and were assayed by heteroduplex analysis, to identify polymorphisms. Two polymorphisms (1333TA/TATA and 2710 C/T) were found and were used with two previously described polymorphisms, to screen a total of 41 families. Twenty-two of these families were shown to be informative (11 for Crouzon syndrome and 11 for Pfeiffer syndrome). Eleven different mutations in the 22 families were detected by either restriction digest or allele-specific oligonucleotide hybridization of ARMS PCR products. We molecularly proved the origin of these different mutations to be paternal for all informative cases analyzed (P=2. 4x10-7; 95% confidence limits 87%-100%). Advanced paternal age was noted for the fathers of patients with Crouzon syndrome or Pfeiffer syndrome, compared with the fathers of control individuals (34. 50+/-7.65 years vs. 30.45+/-1.28 years, P<.01). Our data on advanced paternal age corroborates and extends previous clinical evidence based on statistical analyses as well as additional reports of advanced paternal age associated with paternal origin of three sporadic mutations causing Apert syndrome (FGFR2) and achondroplasia (FGFR3). Our results suggest that older men either have accumulated or are more susceptible to a variety of germline mutations.

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Figures

Figure 1
Figure 1
Partial map of the human FGFR2 gene. Unblackened arrow indicates the ARMS primer; diagonally hatched arrows, ASOs; blackened arrows, all other primers; vertical arrows, locations of the polymorphisms flanking exons IIIa and IIIc.
Figure 2
Figure 2
Polymorphism detection in three unrelated control individuals. A, 1333TA/TATA polymorphism. Top panels show gel electrophoresis of ARMS PCR products derived from primers 13 and 14 (top left) or from primers 13 and 15 (top right). Bottom panels show PCR products that were amplified with primers 5 and 6, were dotted onto filters, and were hybridized with either ASO 7 (bottom left) or ASO 8 (bottom right). Lane 1 represents the heterozygous individual; lane 2, the individual that is homozygous for the TATA polymorphism; lane 3, the individual that is homozygous for the TA polymorphism. B, 2710C/T polymorphism. Top panels show gel electrophoresis of PCR products amplified with the use of primers 9 and 10. Bottom panels show the same PCR products dotted onto filters and hybridized with either ASO 12 (bottom left) or ASO 11 (bottom right). No ARMS PCR products are shown. Lane 1 represents the heterozygous individual; lane 2, the individual that is homozygous for the T polymorphism; lane 3, the individual that is homozygous for the C polymorphism.
Figure 3
Figure 3
Determination of the origin of mutation by means of ARMS PCR followed by restriction digest. A, top panel, primers 13 and 14 were used to amplify 889 bp, starting from the TA variant and extending 3′ to exon IIIc (in which the mutation is located). Unmarked lane indicates size markers φX174 DNA digested with HaeIII. Templates for ARMS PCR are as follows: affected child (subject 1580) (lane C), unaffected father (subject 1587) (lane F), and unaffected mother (subject 1586) (lane M). A, bottom panel, primers 13 and 15 were used to amplify 891 bp from the TATA variant to 3′ of the exon IIIc. Templates for ARMS PCR are the same as those for the top panel. B, The G→A nucleotide change for the C342Y mutation creates an RsaI restriction site in the patient. Top panel indicates digest of ARMS PCR products amplified with the use of primers 13 and 14. Bottom panel indicates digest of ARMS PCR products amplified with the use of primers 13 and 15. Samples are as follows: affected child, undigested (left lane C); affected child, digested (right lane C); unaffected father, digested (lane F); and unaffected mother, digested (lane M). The arrow indicates the smaller digested product from the child’s allele containing the TA polymorphism.
Figure 4
Figure 4
Determination of the origin of mutation by means of ARMS PCR followed by ASO hybridization. A, ARMS PCR products spanning the region containing the TA/TATA polymorphism and exon IIIc, which contains the mutation. Templates for PCR: affected child (subject 1839) (lane C), unaffected father (subject 1841) (lane F), and unaffected mother (subject 1840) (lane M). Top lanes were PCR amplified with the use of primers 13 and 15, which recognize the TATA polymorphism. Bottom lanes were PCR amplified with the use of primers 13 and 14, which recognize the TA polymorphism. B, diagram of the filter dotted with ARMS PCR products in the same orientation that is seen in A. C, Left filter is hybridized with ASO (GGGTAATTCTATTGGGAT), which recognizes the normal allele. Right filter is hybridized with ASO (GGGTAATTGTATTGGGAT), which recognizes the C→G nucleotide change in the S347C mutation in exon IIIc.
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References

Electronic-Database Information

    1. CEPH, http://www.cephb.fr/
    1. GenBank, http://www.ncbi.nlm.nih.gov/Genbank/ (for oligonucleotide primers and nucleotide numbers [accession number AF169399])
    1. Online Mendelian Inheritance in Man (OMIM), http://www.ncbi.nlm.nih.gov/Omim/ (for Crouzon syndrome [MIM 123500] and Pfeiffer syndrome [MIM 101600])

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