Gain-of-function amino acid substitutions drive positive selection of FGFR2 mutations in human spermatogonia

Abstract

Despite the importance of mutation in genetics, there are virtually no experimental data on the occurrence of specific nucleotide substitutions in human gametes. C>G transversions at position 755 of FGF receptor 2 (FGFR2) cause Apert syndrome; this mutation, encoding the gain-of-function substitution Ser252Trp, occurs with a birth rate elevated 200- to 800-fold above background and originates exclusively from the unaffected father. We previously demonstrated high levels of both 755C>G and 755C>T FGFR2 mutations in human sperm and proposed that these particular mutations are enriched because the encoded proteins confer a selective advantage to spermatogonial cells. Here, we examine three corollaries of this hypothesis. First, we show that mutation levels at the adjacent FGFR2 nucleotides 752-754 are low, excluding any general increase in local mutation rate. Second, we present three instances of double-nucleotide changes involving 755C, expected to be extremely rare as chance events. Two of these double-nucleotide substitutions are shown, either by assessment of the pedigree or by direct analysis of sperm, to have arisen in sequential steps; the third (encoding Ser252Tyr) was predicted from structural considerations. Finally, we demonstrate that both major alternative spliceforms of FGFR2 (Fgfr2b and Fgfr2c) are expressed in rat spermatogonial stem cell lines. Taken together, these observations show that specific FGFR2 mutations attain high levels in sperm because they encode proteins with gain-of-function properties, favoring clonal expansion of mutant spermatogonial cells. Among FGFR2 mutations, those causing Apert syndrome may be especially prevalent because they enhance signaling by FGF ligands specific for each of the major expressed isoforms.

Fig. 1.

Structural features of human FGFR2 and sequence context around nucleotide 755G. (a) Cartoon of partial gene structure showing exons IIIa, IIIb, IIIc (encoding the extracellular IgIII domain), and TM (encoding the transmembrane segment). The positions of the 749-112G/A SNP, nucleotides 755C in exon IIIa, and 943G in exon IIIc are indicated. Brackets enclose positions of restriction sites AvaI and EcoRV, diagnostic for exons IIIb and IIIc, respectively, in the rat Fgfr2 ortholog. Alternative splicing to generate the spliceforms FGFR2b and FGFR2c is shown above. (b) DNA sequence around the start of exon IIIa (intron sequence is in lowercase and exon sequence is in capitals). Nucleotides are clustered into codons with the corresponding encoded amino acids. The box encloses the MboI restriction site (nucleotides 752-755) used to select for mutant sequences. Below, all 12 possible single-nucleotide substitutions (bold) at the MboI site are shown with the encoded amino acid in brackets. Only the 755C>G (Apert) and 755C > T (normal/Crouzon) mutations have been observed in the heterozygous state in humans.

Fig. 2.

Quantification of mutations at the 752-755 MboI site of FGFR2. (a) Estimated levels of all 12 possible single-nucleotide mutations (see Fig. 1b) in 95 sperm and 11 blood samples (shown separately), arranged in order of decreasing level of total mutations at position 755. (b) Pie chart showing overall proportion of total mutations at each nucleotide in sperm (Left) and blood (Right). The areas of the two circles are proportional to the overall mutation levels from the two sources.

Fig. 3.

Identification of double-mutation events in FGFR2.(a) A sporadic case of Apert syndrome (subject 204F2). (Top) DNA sequencing of FGFR2 showing heterozygosity for the 755_756CG>TC mutation in the proband (Right). Only the 755C>T mutation is present in her father (Left). (Middle) The proband manifests the typical facial appearance and syndactyly of Apert syndrome (Right), whereas her father has clinically normal hands (Left). (Bottom) Family pedigree, showing patterns of digestion of FGFR2 exon IIIa with DpnII (an isoschizomer of MboI) and HinfI. The specific HinfI digestion product in the proband (black circle) indicates that the two substitutions identified on DNA sequencing are present on the same allele. WT, normal control. (b) Identification of a Ser252Tyr mutation in the sperm of a 60-year-old man. (Upper) Pyrogram from the donor's unspiked sperm sample. E and S denote addition of enzyme and substrate, respectively. The fifth (A) and sixth (G) nucleotide dispensations (green arrows) normally yield peaks of equal height; the absence of a peak at the sixth dispensation indicates that in this sample, 755C>A mutations are not followed by 756G. (Lower) Sequencing of a mutant clone from this PCR product reveals the double-mutation 755_756CG>AC (arrowheads). The pyrogram shows that this mutation is present at comparable levels to 755G (black arrow) and 755T (red arrows). (c) Measurement of levels of the mutations 755C>G (Left) and 755C>T (Right) in two sperm samples from a 49-year-old man heterozygous for the 943G>T (Ala315Ser) mutation. Red triangles show levels for this individual (mean ± 95% equal tail probability interval) normalized with respect to previous measurements on 99 normal donors (white circles) (17).

Fig. 4.

Expression of Fgfr2b and Fgfr2c spliceforms in two rat spermatogonial stem cell lines. Digestion of RT-PCR products with AvaI (specific for Fgfr2b) and EcoRV (specific for Fgfr2c) compared with uncut product and a DNA-negative control (-), for cDNA from cell lines GC-5spg (Left) and GC-6spg (Right). The product from GC-5spg digests completely with EcoRV, indicating that only the Fgfr2c spliceform is present. By contrast, the majority of product from GC-6spg digests with AvaI, indicating predominance of Fgfr2b; the faint EcoRV digestion product shows that a minority of Fgfr2c is also present.

Fig. 5.

Diagrammatic representation of mutation and selection processes in spermatogonial cells, leading to differing prevalence of various FGFR2 mutations. Pairs of parallel lines denote the two FGFR2 alleles with the identity of nucleotides at positions 755 and 943 (d only) specifically indicated. Radiating clusters of arrowed lines represent independent mutational events with the number of arrows reflecting relative mutation frequency. Gray sectors indicate expansion over time of mutant clones bearing the indicated genotype. (a) 755C>G arising in a WT individual: the C>G transversion is relatively infrequent, but the Ser252Trp substitution confers a strong selective advantage. (b) 755C>T arising in a WT individual: C>T transitions occur frequently at this CpG dinucleotide, but the Ser252Leu substitution confers a weak selective advantage compared with Ser252Trp, leading to slower clonal expansion. (c) 756G>C arising in the spermatogonial cell of an individual constitutionally heterozygous for 755C>T. Mutations arising on 755T alleles encode Ser252Phe; selective advantage is comparable to Ser252Trp. Mutations arising on 755C alleles are synonymous and selectively neutral. Note that the processes illustrated in b and c, if they occurred sequentially in a single spermatogonial cell, would lead to double mutations in the sperm of WT individuals. (d) 755C>T arising in an individual constitutionally heterozygous for 943G>T. Mutations on 943T alleles encode the synergistic double-substitution Ser252Leu; Ala315Ser in cis, leading to strong selective advantage. Mutations arising on the WT (943G) allele are more weakly selected as in b.