Fig 1.
Inclusion/exclusion criteria flowchart.
We identified 54 patients with PHP1A and GH deficiency who had brain CT and/or MRI imaging within our cohort. Of these, 47 had a brain MRI performed. One participant was excluded, as we were unable to confirm the imaging data. This resulted in 46 participants for the prevalence calculations. From these, 3 were further excluded, as they were either too young for evaluation of GH status or had a brain MRI >2 years prior to GH stimulation testing. The remaining 43 participants were included in the GH versus CM1/LLCT correlation calculations.
Table 1.
Characteristics of mutation-confirmed PHP1A cohort.
Fig 2.
Brain MRI of PHP1A participant.
Sagittal MRI image from participant #5. The cerebellar tonsils extend 7 mm below the foramen magnum reflecting Chiari type 1 malformation.
Fig 3.
AHO mouse model generated by heterozygous inactivation of Gnas exon 1 (Gnas E1+/-) displays cranial hypoplasia.
(A, B) Representative x-ray images of the skull of (A) 3 week and (B) 12-week-old WT, Gnas E1+/-p and Gnas E1+/-m mice. (C) Representative three dimensional reconstructions of the skull of 12-week-old WT, Gnas E1+/-p and Gnas E1+/-m mice. (D) Measurements of total skull length demonstrate that both Gnas E1+/-p and Gnas E1+/-m mice display a reduction in skull length at both 3 and 12 weeks of age when compared to WT. (E) Measurement of total skull height demonstrate no significant variations between WT and Gnas E1+/- mice at 3 weeks of age. Gnas E1+/-m mice at 12 weeks exhibited a mild but statistically significant increase in skull height when compared to WT mice. (F) Measurement of cranial dome angle demonstrates that both 12-week-old Gnas E1+/-m and Gnas E1+/-p mice exhibit significantly increased cranial dome angles when compared to WT mice. Sample size per genotype per experiment is listed on each graph. All statistical tests were performed by ANOVA with post-hoc Tukey tests for multiple comparisons, and p-values are displayed for each comparison.
Fig 4.
Gnas E1+/- mice do not display significant variations in the rate of interparietal or lambdoid suture closure.
(A) Representative whole mount images of the cranium of 2-week-old WT, Gnas E1+/-p and Gnas E1+/-m mice. (B-D) Representative images of the cranium of 1-week- old (B) WT, (C) Gnas E1+/-p and (D) Gnas E1+/-m mice stained with calcein blue to visualize total mineral content and distance of interparietal and lambdoid sutures. (E-F) Quantification of the (E) Interparietal and (F) Lambdoid suture length demonstrated no significant differences between 1 week-old WT and Gnas E1+/- mice. Sample size per genotype per experiment is listed on each graph. All statistical tests were performed using a two-way ANOVA with post-hoc Tukey test for multiple comparisons, and p-values are displayed for each comparison.
Fig 5.
Gnas E1+/- mice display a significant reduction in spheno-occipital synchondrosis length.
Representative low power (A-C) and higher power (D-F) whole mount images of the cranial base of 2-week-old (P14) (A, D) WT, (B, E) Gnas E1+/-p and (C, F) Gnas E1+/-m mice. Dashed rectangle in A-C is shown as larger image within D-F and highlights spheno-occipital synchondrosis. (G-I) Representative images of the cranial base of 1-week-old (P7) (G) WT, (H) Gnas E1+/-p and (I) Gnas E1+/-m mice stained with safranin O and fast green. (J-L) Representative images of the cranial base of 1-week-old (P7) (J) WT, (K) Gnas E1+/-p and (L) Gnas E1+/-m mice stained with safranin O and fast green. (M) Measurement of total SOS length (yellow line) among WT, Gnas E1+/-p and Gnas E1+/-m mice at both one and two weeks of age. Sample size per genotype per experiment is listed on each graph. All statistical tests completed using a two-way ANOVA with post-hoc Tukey test for multiple comparisons, and p-values are displayed for each comparison. For orientation, the sphenoid bone (SB) and occipital bone (OB) are identified on panels D-F for appropriate localization in relation to the SOS.
Fig 6.
Accelerated reduction in spheno-occipital synchondrosis length within E1+/- mice is associated with a reduction in the number of proliferating chondrocytes.
(A-F) Representative images of the SOS (yellow line) in (A-C) 1-week-old mice and (D-F) 2-week-old mice. In Fig 6 (A, D) are WT, (B, E) are Gnas E1+/-p, and (C, F) are Gnas E1+/-m mice stained for Alkaline phosphatase (red), EdU (green), and DAPI (blue). (G) Quantification of the percentage of EdU+ chondrocytes to the total number of chondrocytes within the resting and proliferative zone at 1 week of age demonstrates that both Gnas E1+/-p and Gnas E1+/-m mice display a significant reduction in proliferating chondrocytes compared to WT. No significant differences were observed at 2 weeks of age. (H) Quantification of the resting and proliferative zone lengths (defined as ALP negative zone of synchondrosis) (blue line) were significantly reduced in both Gnas E1+/-p and Gnas E1+/-m mice when compared to WT at 1 week of age. No significant differences were observed at 2 weeks of age. (I) Quantification of the hypertrophic zone length (defined as ALP+ zone of synchondrosis) (orange lines) demonstrated no significant differences between WT and Gnas E1+/- mice at 1 or 2 weeks of age. Sample size per genotype per experiment is listed on each graph. All statistical tests were completed using a two-way ANOVA with post-hoc Tukey test for multiple comparisons, and p-values are displayed for each comparison.
Fig 7.
Gnas E1+/-m mice exhibit a reduction in foramen magnum width and area.
(A, B) Representative three-dimensional reconstructions of the (A) basioccipital bone and (B) foramen magnum of 12-week old WT, Gnas E1+/-p and Gnas E1+/-m mice. (C) Measurements of basioccipital bone length in 12-week WT and Gnas E1+/- mice demonstrate no significant differences. (D) Measurement of foramen magnum width, height, and area demonstrate that Gnas E1+/-m and Gnas E1+/-p mice display no significant differences in foramen magnum width, height or total area when compared to WT mice. Sample size per genotype per experiment is listed on each graph. All statistical tests were completed using ANOVA with post-hoc Tukey test for multiple comparisons, and p-values are displayed for each comparison.
Fig 8.
Gnas E1+/-m mice display cranial hyperostosis and enhanced calvarial bone formation in vivo.
(A) Representative 3D reconstruction of the cranial vault of 12-week WT, Gnas E1+/-p, and Gnas E1+/-m mice demonstrating hyperostosis within Gnas E1+/-m mice. (B) Representative calcein and alizarin complexone double labeling on the calvaria of 12-week WT, Gnas E1+/-p, and Gnas E1+/-m mice. (C-E) Quantification of (C) Bone formation rate (BFR); (D) mineralizing surface to bone surface (MS/BS); and (E) Mineral apposition rate (MAR) within the calvaria demonstrates Gnas E1+/-m mice display enhanced bone formation when compared to both WT and Gnas E1+/-p mice. No significant differences were observed between WT and Gnas E1+/-p mice. Sample size per genotype per experiment is listed on each graph. All statistical tests were completed using ANOVA with post-hoc Tukey test for multiple comparisons, and p-values are displayed for each comparison.