Genotype-Phenotype Correlation of Tracheal Cartilaginous Sleeves and Fgfr2 Mutations in Mice

Abstract

Objectives: To characterize tracheal cartilage morphology in mouse models of fibroblast growth factor receptor (Fgfr2)-related craniosynostosis syndromes. To establish relationships between specific Fgfr2 mutations and tracheal cartilaginous sleeve (TCS) phenotypes in these mouse models.

Methods: Postnatal day 0 knock-in mouse lines with disease-specific genetic variations in the Fgfr2 gene (Fgfr2^C342Y/C342Y , Fgfr2^C342Y/+ , Fgfr2^+/Y394C , Fgfr2^+/S252W , and Fgfr2^+/P253R ) as well as line-specific controls were utilized. Tracheal cartilage morphology as measured by gross analyses, microcomputed tomography (μCT), and histopathology were compared using Chi-squared and single-factor analysis of variance statistical tests.

Results: A greater proportion of rings per trachea were abnormal in Fgfr2^C342Y/+ tracheas (63%) than Fgfr2^+/S252W (17%), Fgfr2^+/P253R (17%), Fgfr2^+/Y394C (12%), and controls (10%) (P < .001 for each vs. Fgfr2^C342Y/+ ). TCS segments were found only in Fgfr2^C342Y/C342Y (100%) and Fgfr2^C342Y/+ (72%) tracheas. Cricoid and first-tracheal ring fusion was noted in all Fgfr2^C342Y/C342Y and 94% of Fgfr2^C342Y/+ samples. The Fgfr2^C342Y/C342Y and Fgfr2^C342Y/+ groups were found to have greater areas and volumes of cartilage than other lines on gross analysis and μCT. Histologic analyses confirmed TCS among the Fgfr2^C342Y/C342Y and Fgfr2^C342Y/+ groups, without appreciable differences in cartilage morphology, cell size, or density; no histologic differences were observed among other Fgfr2 lines compared to controls.

Conclusion: This study found TCS phenotypes only in the Fgfr2^C342Y mouse lines. These lines also had increased tracheal cartilage compared to other mutant lines and controls. These data support further study of the Fgfr2 mouse lines and the investigation of other Fgfr2 variants to better understand their role in tracheal development and TCS formation.

Level of evidence: NA Laryngoscope, 131:E1349-E1356, 2021.

Keywords: Apert syndrome; Beare-Stevenson syndrome; Crouzon syndrome; FGFR2; craniosynostosis; tracheal anomalies; tracheal cartilaginous sleeve.

Figure 1:

Tracheal ring types, their assigned codes, descriptions and frequencies at which they were observed in control specimens. “Normal” tracheal ring types (frequency in controls ≥ 20%) are highlighted in green whereas “Abnormal” and “Sleeve-Type” are highlighted in yellow and red, respectively. Note: Sleeve-type segments are classified both as “Abnormal” and “Sleeve-Type” for analyses.

Figure 2:

Panels A-F represent alcian blue stained whole-mount specimens, A) Fgfr2^C342Y/C342Y, B) Fgfr2^C342Y/+, C) Fgfr2^+/Y394C, D) Fgfr2^+/S252W, E) Fgfr2^+/P253R and F) Fgfr2^+/+. Scale bars = 1.0 mm. Panels G and H demonstrate box plots representing the proportion of tracheal rings that were abnormal and cartilage area, respectively. Center lines show the medians; box limits indicate the 25th and 75th percentiles; whiskers extend 1.5 times the interquartile range from the 25th and 75th percentiles; plus signs represent sample means; data points are plotted as open circles. ** p < .01 vs Fgfr2^C342Y/C342Y *** p < .001 vs Fgfr2^C342Y/C342Y ††† p < .001 vs Fgfr2^C342Y/+ ‡ p < .05 vs Fgfr2^+/P253R

Figure 3:

Panels A-F represent 3D-reconstructions of microcomputed-tomography specimens, A) Fgfr2^C342Y/C342Y, B) Fgfr2^C342Y/+, C) Fgfr2^+/Y394C, D) Fgfr2^+/S252W, E) Fgfr2^+/P253R and F) Fgfr2^+/+. Scale bars = 1.0 mm. Panels G and H demonstrate box and whisker plots representing cartilage volume and airway volume, respectively. Center lines show the medians; box limits indicate the 25th and 75th percentiles; whiskers extend 1.5 times the interquartile range from the 25th and 75th percentiles; crosses represent sample means; data points are plotted as open circles. * p < .05 vs Fgfr2^C342Y/C342Y *** p < .001 vs Fgfr2^C342Y/C342Y ††† p < .001 vs Fgfr2^C342Y/+

Figure 4:

Representative images of Movat pentachrome stain (panels A, B) and hematoxylin & eosin stain (panels C, D) of Fgfr2^+/+ (panels A, C), and Fgfr2^C342Y/C342Y (panels B, D) highlight cartilage (stained green) and mitotic figures (arrows). Panel E shows a bar graph representing the number of chondrocytes per area of hyaline cartilage as assessed on 5 random images taken at 40x for 2 samples per group. Error bars represent standard deviations. Scale bars = 50 μm for panels A, B, and 25 μm for panels C, D.