Prevalence of Chiari malformation type 1 is increased in pseudohypoparathyroidism type 1A and associated with aberrant bone development

Abstract

Background: Albright hereditary osteodystrophy (AHO) is caused by heterozygous inactivating mutations in GNAS. Patients with maternally-inherited mutations develop pseudohypoparathyroidism type 1A (PHP1A) with multi-hormone resistance and aberrant craniofacial and skeletal development among other abnormalities. Chiari malformation type 1 (CM1), a condition in which brain tissue extends into the spinal canal when the skull is too small, has been reported in isolated cases of PHP1A. It has been hypothesized to be associated with growth hormone (GH) deficiency. Given the adverse clinical sequelae that can occur if CM1 goes unrecognized, we investigated the previously undetermined prevalence of CM1, as well as any potential correlations with GH status, given the known increased prevalence of GH deficiency in PHP1A. We also investigated these metrics for low lying cerebellar tonsils (LLCT), defined as tonsillar descent less than 5 mm below the foramen magnum. In addition, we investigated possible correlations of CM1/LLCT with advanced hand/wrist bone ages and craniofacial abnormalities known to occur in PHP1A to determine whether premature chondrocyte differentiation and/or aberrant craniofacial development could be potential etiologies of CM1/LLCT through both human studies and investigations of our AHO mouse model.

Methods: We examined patients with PHP1A in our clinic and noticed CM1 more frequently than expected. Therefore, we set out to determine the true prevalence of CM1 and LLCT in a cohort of 54 mutation-confirmed PHP1A participants who had clinically-indicated brain imaging. We examined potential correlations with GH status, clinical features, biological sex, genotype, and hand/wrist bone age determinations. In addition, we investigated the craniofacial development in our mouse model of AHO (Gnas E1+/-m) by histologic analyses, dynamic histomorphometry, and micro-computerized tomographic imaging (MCT) in order to determine potential etiologies of CM1/LLCT in PHP1A.

Results: In our cohort of PHP1A, the prevalence of CM1 is 10.8%, which is at least 10-fold higher than in the general population. If LLCT is included, the prevalence increases to 21.7%. We found no correlation with GH status, biological sex, genotype, or hand/wrist bone age. Through investigations of our Gnas E1+/-m mice, the correlate to PHP1A, we identified a smaller cranial vault and increased cranial dome angle with evidence of hyperostosis due to increased osteogenesis. We also demonstrated that there was premature closure of the spheno-occipital synchondrosis (SOS), a cartilaginous structure essential to the development of the cranial base. These findings lead to craniofacial abnormalities and could contribute to CM1 and LLCT development in PHP1A.

Conclusion: The prevalence of CM1 is at least 10-fold higher in PHP1A compared to the general population and 20-fold higher when including LLCT. This is independent of the GH deficiency that is found in approximately two-thirds of patients with PHP1A. In light of potential serious consequences of CM1, clinicians should have a low threshold for brain imaging. Investigations of our AHO mouse model revealed aberrant cranial formation including a smaller cranium, increased cranial dome angle, hyperostosis, and premature SOS closure rates, providing a potential etiology for the increased prevalence of CM1 and LLCT in PHP1A.

Conflict of interest statement

The authors have declared that no competing interests exist.

Copyright: © 2023 Krishnan et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.

Figures

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.

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.