Selfish mutations dysregulating RAS-MAPK signaling are pervasive in aged human testes

Abstract

Mosaic mutations present in the germline have important implications for reproductive risk and disease transmission. We previously demonstrated a phenomenon occurring in the male germline, whereby specific mutations arising spontaneously in stem cells (spermatogonia) lead to clonal expansion, resulting in elevated mutation levels in sperm over time. This process, termed "selfish spermatogonial selection," explains the high spontaneous birth prevalence and strong paternal age-effect of disorders such as achondroplasia and Apert, Noonan and Costello syndromes, with direct experimental evidence currently available for specific positions of six genes (FGFR2, FGFR3, RET, PTPN11, HRAS, and KRAS). We present a discovery screen to identify novel mutations and genes showing evidence of positive selection in the male germline, by performing massively parallel simplex PCR using RainDance technology to interrogate mutational hotspots in 67 genes (51.5 kb in total) in 276 biopsies of testes from five men (median age, 83 yr). Following ultradeep sequencing (about 16,000×), development of a low-frequency variant prioritization strategy, and targeted validation, we identified 61 distinct variants present at frequencies as low as 0.06%, including 54 variants not previously directly associated with selfish selection. The majority (80%) of variants identified have previously been implicated in developmental disorders and/or oncogenesis and include mutations in six newly associated genes (BRAF, CBL, MAP2K1, MAP2K2, RAF1, and SOS1), all of which encode components of the RAS-MAPK pathway and activate signaling. Our findings extend the link between mutations dysregulating the RAS-MAPK pathway and selfish selection, and show that the aging male germline is a repository for such deleterious mutations.

Figure 1.

Distribution of validated variants in testis slices 1D (A), 2F (B), 4B (C), and 5J (D). Testicular biopsy numbers are located to the left of each testis slice. Some biopsies were further dissected into two pieces of which the orientation is unknown; these are indicated with a diagonal dashed line (e.g., Tes2F 30a,b). Each variant has a distinct number (as listed in Table 1) and is colored according to gene—FGFR2 (purple), FGFR3 (orange), KRAS (black), PTPN11 (blue), RET (pink), and newly associated gene (red)—and is also indicated on the figure key. The size of each circle is proportional to the observed variant allele frequency (VAF) in each biopsy as indicated by black dots on the figure key. Identical variants in different biopsies have been connected by lines that likely track the seminiferous tubule trajectory across the testis and therefore may represent a single “clonal event”; note that the path of the clone has been arbitrarily drawn and may not represent the true trajectory of the tubule. Dark gray segments represent biopsies that were not sequenced due to insufficient material quality/quantity (see Methods). Light gray segments represent nontubular regions of tissue. The age of the individual from whom the testis was collected is indicated on the figure (for further details on the testicular samples, see Supplemental Table S5). The remaining five slices of Tes4 are presented in Supplemental Figure S2. Tes3D is omitted as no variants were identified. Variants are numbered in order of tier: Tier 1 (1–39), Tier 2 (40–57), Tier 3 (58–61). Letters in brackets refer to variants associated with germline disorders [G] and/or reported in the COSMIC database [C]; for further details, see also Table 1 and Supplemental Table S3.

Figure 2.

Spontaneous mutations in FGFR2 (A) and PTPN11 (also known as SHP2; B) identified in testicular biopsies. (A, I) Ten validated variants positioned along the amino acid sequence of FGFR2 (x-axis, see panel V), ranging in VAF from 0.06% to 2.95% (y-axis), identified in Tes1D, Tes2F, and Tes4. Numbers correspond to those in Table 1; two different variants (c.870G > C or T) predicted to cause the same p.Trp290Cys substitution (nos. 11, 12) were identified. (II) Relative location and length of amplicons used to sequence main hotspots of FGFR2 are plotted on the x-axis. Median coverage per amplicon is plotted on the y-axis. All amplicons had median coverage above the cut-off (red dashed line) of 5000×. (III) Number of reported constitutional variants encoding amino acid substitutions in FGFR2 associated with developmental disorders (sqrt scale) (updated from Wilkie 2005). (IV) Number of reported somatic amino acid substitutions in FGFR2 in cancer (COSMIC v82). (V) Protein domains of FGFR2. Annotations and protein structure are based on transcript ID NM_000141 and Uniprot ID P21802 (v2017_01), respectively. (B, I) Twenty validated variants positioned along the amino acid sequence of SHP2 (x-axis, see panel V), ranging in VAF from 0.09% to 1.02% (y-axis), identified in Tes1D, Tes2F, and Tes4. (II) Location and size of amplicons used to sequence main hotspots of PTPN11 are plotted on the x-axis. Median coverage per amplicon is plotted on the y-axis. All amplicons except one had median coverage above the cut-off of 5000×. (III) Number of reported constitutional variants encoding amino acid substitutions in SHP2 associated with developmental disorders (sqrt scale). (IV) Number of reported somatic amino acid substitutions in SHP2 in cancer (COSMIC v82). (V) Protein domains of SHP2. Annotations and protein structure are based on transcript ID NM_002834 and Uniprot ID Q06124 (v2017_01), respectively.

Figure 3.

Visualization of mutant tubules in Testis 2. (A) A 5-µm-thick section from Tes2E, a FFPE block of tissue adjacent to the testis slice 2F (B), immunostained with anti-MAGEA4 antibody to label spermatogonia. Seminiferous tubules with enhanced MAGEA4 immunopositivity, suggestive of the presence of mutant clones are labeled with small red pins and boxed. Scale bar, 5 mm. (C,D) High-magnification view of cross-sections with MAGEA4-enhanced immunopositivity in two localized areas are labeled with dotted lassoes representing the laser-microdissected regions. Scale bars, 100 µm. (E,F) Results from targeted resequencing of the microdissected seminiferous tubules labeled by dotted lassoes in C and D, respectively, viewed in integrated genome viewer (IGV), with local genomic sequence context indicated at the bottom. VAF of mutant reads is indicated on the top using color specific for each base pair; spontaneous pathogenic FGFR2 c.755C > G (no. 7; E) and PTPN11 c.214G > C (no. 25; F) variants were identified in DNA extracted from microdissected tubule cross-sections, but not in DNA from the whole-tissue section. Comparison of the MAGEA4 section (A) with adjacent testis slice 2F from the RainDance screen (B; the same image as in Figure 1B but showing only the targeted FGFR2 and PTPN11 mutations) shows that both variants match to a mutation previously identified in the corresponding position of testis slice 2F.