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Fig. 3
Genetic analysis of LRRC50 in human seminoma samples.
(A) LRRC50 spans 12 exons on chromosomal arm band 16q24.1. All recovered genetic variations (see Table 2) are indicated in the LRRC50 structure. (B) Chromatogram of SE14-PBL reveals a germline heterozygous nonsense c.1462C>T/p.Arg488* (TMP_ESP_16_84203896) mutation (red arrow in A). Bilateral SE14-seminomas (one sample shown) show a constitutively stronger mutant peak. Analysis of up- and downstream SNPs in closest proximity (blue arrows in A), c.1178A>G/rs17856705 in exon 8 and c.1898T>C/rs2288020 in exon 11, render equivalent peak intensities, indicative of a regional LOH in the tumor. (C) IHC for LRRC50 stains SPG and spermatocytes in normal testis, interestingly; structures reminiscent of cilia in SPG are observed. The SE14 tumor shows complete loss of LRRC50 protein expression. Motile cilia staining of non-tumorigenic tissue (rete testis) from SE14 supports antibody specificity and proper fixation of tumor SE14 tissue. Scale bars; 50 µm. (D) IF of normal testis tissue indicates LRRC50 in cilia on germ cells lining the seminiferous tubule (red arrows) and somatic tissue of the seminiferous tubules (white arrows and insert). Early germ cells localize peripheral to the seminiferous tubule and higher resolution images (maximal projections of Z-stacks) show the presence of solitary cilia in these germ cells in more detail using cilia markers acetylated-α-tubulin (Ac-tub) and detyrosinated-tubulin, LRRC50 locates to cilia in these SPG. Scale bars; 10 μm (E) fTGCT6 and SE14 proband pedigrees. (F) Genotyping LRRC50 in the seminoma collection (n = 38) identified one sample with a c.1769C>T/p.Thr590Met (rs34777958) allele. Additionally, heterozygous variation c.919C>G/p.Gln307Glu (rs111472069) was identified in five seminoma samples, representing a significant enrichment (P = 0.0013). Both variations are absent in a healthy control group (n = 100).