Visualizing the origins of selfish de novo mutations in individual seminiferous tubules of human testes

Abstract

De novo point mutations arise predominantly in the male germline and increase in frequency with age, but it has not previously been possible to locate specific, identifiable mutations directly within the seminiferous tubules of human testes. Using microdissection of tubules exhibiting altered expression of the spermatogonial markers MAGEA4, FGFR3, and phospho-AKT, whole genome amplification, and DNA sequencing, we establish an in situ strategy for discovery and analysis of pathogenic de novo mutations. In 14 testes from men aged 39-90 y, we identified 11 distinct gain-of-function mutations in five genes (fibroblast growth factor receptors FGFR2 and FGFR3, tyrosine phosphatase PTPN11, and RAS oncogene homologs HRAS and KRAS) from 16 of 22 tubules analyzed; all mutations have known associations with severe diseases, ranging from congenital or perinatal lethal disorders to somatically acquired cancers. These results support proposed selfish selection of spermatogonial mutations affecting growth factor receptor-RAS signaling, highlight its prevalence in older men, and enable direct visualization of the microscopic anatomy of elongated mutant clones.

Keywords: germline; mutation; selfish selection; seminiferous tubule; testis.

Fig. 1.

Seminiferous tubules strongly immunopositive for MAGEA4 contain pathogenic mutations. (A–C) Thin sections from three FFPE testes showing spermatogonia, marked by MAGEA4 positivity (brown staining), present in a single layer at the periphery of normal tubular cross-sections (green surround or unlabeled). A subset of tubules (immunopositive tubules) display enhanced MAGEA4 staining (blue surround) due to dense clustering of spermatogonia with strong immunoreactivity. Dideoxy-sequencing traces were obtained from non-WGA DNA extracted from microdissected tissue of an adjacent section. (A) Heterozygous FGFR3 c.1118A>G (p.Y373C) mutations (*) are present in immunopositive tubules, but not in neighboring normal tubules. Clusters of mutation-positive tubules likely represent cross-sections of a single convoluted tubule. (B) In a longitudinal section of a tubule showing the transition from normal to strongly immunopositive staining, the heterozygous HRAS c.37C>G (p.G13R) mutation (*) was specific to the immunopositive portion, pinpointing the boundary between nonmutant and mutant cells. (C) Heterozygous FGFR2 c.758C>G (p.P253R) mutation (*) in immunopositive tubule. (Scale bars: 100 µm.)

Fig. 2.

Independent mutations can populate adjacent MAGEA4 immunopositive tubules, and extensive regional spread can occur. (A) Mutually exclusive FGFR3 c.742C>T (p.R248C) (blue surround) and PTPN11 c.215C>T (p.A72V) (red surround) mutations (*) in neighboring tubules with similar immunopositive appearance. (B) Low magnification view showing extensive region of tubules with FGFR3 c.1948A>G (p.K650E) mutation (*) (blue surround). Immunopositive tubules outside this region harbor a different nucleotide substitution, FGFR2 c.870G>T (p.W290C) (*) (red surround). (Scale bars: 1 mm.)

Fig. 3.

Analysis of spermatogenesis in immunopositive tubules. The difference in Johnsen scores between paired immunopositive and adjacent normal tubules is plotted for each pair; Johnsen scores range between 1 (no seminiferous epithelium) and 10 (full spermatogenesis). In pairwise analysis, the Johnsen score is significantly lower in immunopositive than in normal-staining tubules (P = 5.2 × 10⁻⁵, Wilcoxon signed rank test).

Fig. 4.

Retention of mutant seminiferous tubules in severely atrophic testis. (A) Low magnification view of MAGEA4-stained atrophic testis with the dotted line demarcating the testicular parenchyma. Tubules with spermatogonia (identified by brown stain) are rare and present only in the outlined box. (B) Higher magnification of boxed region, demonstrating that most of the tubular cross-sections containing spermatogonia have an immunopositive appearance (blue surround) and carry the apparently homozygous FGFR3 c.1948A>G (p.K650E) mutation (*). The normally stained tubules (green surround) do not carry the mutation at a detectable level. (Scale bars: 1 mm.)