Exploring FGFR3 Mutations in the Male Germline: Implications for Clonal Germline Expansions and Paternal Age-Related Dysplasias.

Delayed fatherhood results in a higher risk of inheriting a new germline mutation that might result in a congenital disorder in the offspring. In particular, some FGFR3 mutations increase in frequency with age, but there are still a large number of uncharacterized FGFR3 mutations that could be expanding in the male germline with potentially early- or late-onset effects in the offspring. Here, we used digital polymerase chain reaction to assess the frequency and spatial distribution of 10 different FGFR3 missense substitutions in the sexually mature male germline. Our functional assessment of the receptor signaling of the variants with biophysical methods showed that 9 of these variants resulted in a higher activation of the receptor´s downstream signaling, resulting in 2 different expansion behaviors. Variants that form larger subclonal expansions in a dissected postmortem testis also showed a positive correlation of the substitution frequency with the sperm donor's age, and a high and ligand-independent FGFR3 activation. In contrast, variants that measured high FGFR3 signaling and elevated substitution frequencies independent of the donor's age did not result in measurable subclonal expansions in the testis. This suggests that promiscuous signal activation might also result in an accumulation of mutations before the sexual maturation of the male gonad with clones staying relatively constant in size throughout time. Collectively, these results provide novel insights into our understanding of the mutagenesis of driver mutations and their resulting mosaicism in the male germline with important consequences for the transmission and recurrence of associated disorders.

Measurement of FGFR3 signaling at the cell membrane via total internal reflection fluorescence microscopy to compare the activation of FGFR3 mutants.

Fibroblast growth factor receptors (FGFRs) initiate signal transduction via the RAS/mitogen-activated protein kinase pathway by their tyrosine kinase activation known to determine cell growth, tissue differentiation, and apoptosis. Recently, many missense mutations have been reported for FGFR3, but we only know the functional effect for a handful of them. Some mutations result in aberrant FGFR3 signaling and are associated with various genetic disorders and oncogenic conditions. Here, we employed micropatterned surfaces to specifically enrich fluorophore-tagged FGFR3 (monomeric GFP [mGFP]-FGFR3) in certain areas of the plasma membrane of living cells. We quantified receptor activation via total internal reflection fluorescence microscopy of FGFR3 signaling at the cell membrane that captured the recruitment of the downstream signal transducer growth factor receptor-bound 2 (GRB2) tagged with mScarlet (GRB2-mScarlet) to FGFR3 micropatterns. With this system, we tested the activation of FGFR3 upon ligand addition (fgf1 and fgf2) for WT and four FGFR3 mutants associated with congenital disorders (G380R, Y373C, K650Q, and K650E). Our data showed that ligand addition increased GRB2 recruitment to WT FGFR3, with fgf1 having a stronger effect than fgf2. For all mutants, we found an increased basal receptor activity, and only for two of the four mutants (G380R and K650Q), activity was further increased upon ligand addition. Compared with previous reports, two mutant receptors (K650Q and K650E) had either an unexpectedly high or low activation state, respectively. This can be attributed to the different methodology, since micropatterning specifically captures signaling events at the plasma membrane. Collectively, our results provide further insight into the functional effects of mutations to FGFR3.

Pathogenic postzygotic mosaicism in the tyrosine receptor kinase pathway: potential unidentified human disease hidden away in a few cells.

Mutations occurring during embryonic development affect only a subset of cells resulting in two or more distinct cell populations that are present at different levels, also known as postzygotic mosaicism (PZM). Although PZM is a common biological phenomenon, it is often overlooked as a source of disease due to the challenges associated with its detection and characterization, especially for very low-frequency variants. Moreover, PZM can cause a different phenotype compared to constitutional mutations. Especially, lethal mutations in receptor tyrosine kinase (RTK) pathway genes, which exist only in a mosaic state, can have completely new clinical manifestations and can look very different from the associated monogenic disorder. However, some key questions are still not addressed, such as the level of mosaicism resulting in a pathogenic phenotype and how the clinical outcome changes with the development and age. Addressing these questions is not trivial as we require methods with the sensitivity to capture some of these variants hidden away in very few cells. Recent ultra-accurate deep-sequencing approaches can now identify these low-level mosaics and will be central to understand systemic and local effects of mosaicism in the RTK pathway. The main focus of this review is to highlight the importance of low-level mosaics and the need to include their detection in studies of genomic variation associated with disease.

A novel microRNA located in the TrkC gene regulates the Wnt signaling pathway and is differentially expressed in colorectal cancer specimens.

Tropomyosin receptor kinase C (TrkC) is involved in cell survival, apoptosis, differentiation, and tumorigenesis. TrkC diverse functions might be attributed to the hypothetical non-coding RNAs embedded within the gene. Using bioinformatics approaches, a novel microRNA named TrkC-miR2 was predicted within the TrkC gene capable of regulating the Wnt pathway. For experimental verification of this microRNA, the predicted TrkC-premir2 sequence was overexpressed in SW480 cells, which led to the detection of two mature TrkC-miR2 isomiRs, and their endogenous forms were detected in human cell lines as well. Later, an independent promoter was deduced for TrkC-miR2 after the treatment of HCT116 cells with 5-azacytidine, which resulted in differential expression of TrkC-miR2 and TrkC host gene. RT-quantitative PCR and luciferase assays indicated that the APC2 gene is targeted by TrkC-miR2, and Wnt signaling is up-regulated. Also, Wnt inhibition by using small molecules along with TrkC-miR2 overexpression and TOP/FOP flash assays confirmed the positive effect of TrkC-miR2 on the Wnt pathway. Consistently, TrkC-miR2 overexpression promoted SW480 cell survival, which was detected by flow cytometry, MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) assays, and crystal violate analysis. RT-qPCR analysis revealed that TrkC-miR2 is significantly up-regulated (∼70 times) in colorectal tumor tissues compared with their normal pairs. Moreover, the TrkC-miR2 expression level discriminated grades of tumor malignancies, which was consistent with its endogenous levels in HCT116, HT29, and SW480 colorectal cancer cell lines. Finally, an opposite expression pattern was observed for TrkC-miR2 and the APC2 gene in colorectal cancer specimens. In conclusion, here we introduce TrkC-miR2 as a novel regulator of Wnt signaling, which might be a candidate oncogenic colorectal cancer biomarker.