International Journal of Molecular Sciences Article Calvaria Bone Transcriptome in Mouse Models of Osteogenesis Imperfecta Pierre Moffatt 1,2 , Iris Boraschi-Diaz 1,3, Juliana Marulanda 1,3, Ghalib Bardai 1 and Frank Rauch 1,3,* 1 Shriners Hospital for Children-Canada, Montreal, QC H4A 0A9, Canada; [email protected] (P.M.); [email protected] (I.B.-D.); [email protected] (J.M.); [email protected] (G.B.) 2 Department of Pediatrics, McGill University, Montreal, QC H4A 3J1, Canada 3 Department of Human Genetics, McGill University, Montreal, QC H3A 0C7, Canada * Correspondence: [email protected]; Tel.: +1-514-282-7193 Abstract: Osteogenesis imperfecta (OI) is a bone fragility disorder that is usually caused by mutations affecting collagen type I. We compared the calvaria bone tissue transcriptome of male 10-week-old heterozygous Jrt (Col1a1 mutation) and homozygous oim mice (Col1a2 mutation) to their respec- tive littermate results. We found that Jrt and oim mice shared 185 differentially expressed genes (upregulated: 106 genes; downregulated: 79 genes). A total of seven genes were upregulated by a factor of two or more in both mouse models (Cyp2e1, Slc13a5, Cgref1, Smpd3, Ifitm5, Cthrc1 and Rerg). One gene (Gypa, coding for a blood group antigen) was downregulated by a factor of two or more in both OI mouse models. Overrepresentation analyses revealed that genes involved in ‘ossification’ were significantly overrepresented among upregulated genes in both Jrt and oim mice, whereas hematopoietic genes were downregulated. Several genes involved in Wnt signaling and transforming growth factor beta signaling were upregulated in oim mice, but less so in Jrt mice. Thus, Citation: Moffatt, P.; Boraschi-Diaz, this study identified a set of genes that are dysregulated across various OI mouse models and are I.; Marulanda, J.; Bardai, G.; Rauch, F. likely to play an important role in the pathophysiology of this disorder. Calvaria Bone Transcriptome in Mouse Models of Osteogenesis Keywords: osteogenesis imperfecta; RNA sequencing; transforming growth factor beta; Imperfecta. Int. J. Mol. Sci. 2021, 22, Wnt signaling 5290. https://doi.org/10.3390/ ijms22105290 Academic Editor: Alberto Falchetti 1. Introduction Osteogenesis imperfecta (OI) is a heritable connective tissue disorder that is clinically Received: 7 April 2021 characterized by low bone mass, bone fragility, long bone deformity, scoliosis, discolored Accepted: 12 May 2021 Published: 18 May 2021 sclera and dentinogenesis imperfecta [1]. In the large majority of individuals with OI, the condition is caused by mutations in Col1a1 or Col1a2 that directly affect the bone matrix Publisher’s Note: MDPI stays neutral protein collagen type I [2]. However, pathogenic variants in about 20 other genes can also with regard to jurisdictional claims in give rise to an OI phenotype [1]. published maps and institutional affil- How mutations in Col1a1 and Col1a2 lead to low bone mass and bone fragility is iations. not entirely clear. The low bone mass of OI is not simply caused by an inability of the osteoblast system to produce bone. Indeed, many children with severe OI have increased bone formation rates, as shown by iliac bone histomorphometry [3,4]. Low bone mass in OI is therefore likely caused by dysregulation of bone cell activity rather than the incapacity of osteoblasts to produce enough organic bone matrix. Copyright: © 2021 by the authors. Several mouse models with mutations in Col1a1 or Col1a2 are available to study Licensee MDPI, Basel, Switzerland. This article is an open access article the dysregulation of bone cells. A widely used model is the oim mouse that harbors a distributed under the terms and frameshift mutation in Col1a2 [5]. Homozygous oim mice are small and have spontaneous conditions of the Creative Commons fractures, mimicking the manifestations of severe OI in humans. The Jrt mouse is a model Attribution (CC BY) license (https:// of dominant severe OI caused by a splice-site mutation in Col1a1 that leads to an 18-amino creativecommons.org/licenses/by/ acid deletion in the collagen type I alpha 1 chain [6]. Similar to the homozygous oim mouse, 4.0/). the heterozygous Jrt mouse has spontaneous fractures and bone deformities. Int. J. Mol. Sci. 2021, 22, 5290. https://doi.org/10.3390/ijms22105290 https://www.mdpi.com/journal/ijms Int. J. Mol. Sci. 2021, 22, x 2 of 12 Int. J. Mol. Sci. 2021, 22, 5290 2 of 12 acid deletion in the collagen type I alpha 1 chain [6]. Similar to the homozygous oim mouse, the heterozygous Jrt mouse has spontaneous fractures and bone deformities. Thus,Thus, homozygous homozygous oimoim micemice and and heterozygous heterozygous Jrt Jrt mice mice harbor harbor quite quite different different muta- muta- tionstions in in Col1a2Col1a2 andand Col1a1Col1a1,, respectively, butbut shareshare a a similarly similarly severe severe phenotype. phenotype. Identifying Identify- ingmolecular molecular pathways pathways and genesand genes that arethat dysregulated are dysregulated in both in mouse both modelsmouse mightmodels therefore might thereforehelp to delineate help to delineate common common factors that factors are involvedthat are involved in causing in lowcausing bone low mass bone and mass bone andfragility bone infragility the context in the ofcontext mutations of mutations in collagen in collagen type I encoding type I encoding genes. Ingenes. the presentIn the presentstudy, westudy therefore, we therefore performed performed RNA sequencing RNA sequencing in the calvaria in the bone calvaria tissues bone of homozygous tissues of homozygousoim and heterozygous oim and heterozygous Jrt mice and performedJrt mice and differential performed gene differential expression gene analyses. expression We analyses.identified We a setidentified of genes a set that of aregenes dysregulated that are dysregulated across various across OI various mouse OI models mouse and mod- are elslikely and toare play likely an to important play an important role in the role pathophysiology in the pathophysiology of this disorder. of this disorder. 2.2. Results Results ComparedCompared to to wild wild type type (WT) (WT) mice, mice, 231 231 genes genes were were differentially differentially expressed expressed in in Jrt Jrt micemice,, and and 2969 2969 genes genes were were differentially differentially expressed expressed in oim in oim micemice (Figure (Figure 1). 1We). Wefound found 185 genes185 genes that were that differentially were differentially expressed expressed in both inJrt bothand oim Jrt andmice,oim whmice,ich were which all concord- were all antlyconcordantly upregulated upregulated (106 genes) (106 or downregulated genes) or downregulated (79 genes) between (79 genes) the two between models the (Sup- two plementarymodels (Supplementary Tables S1–S6) Tables. S1–S6). FigureFigure 1. 1. ResultsResults of of RNA RNA sequencing sequencing in in calvaria calvaria of of 10 10-week-old-week-old mice. Venn diagramdiagram ofof differentiallydifferen- tiallyexpressed expressed genes genes in oim inand oim Jrtand mice. Jrt mice. TheThe list list of of the the top top 20 20 upregulated upregulated genes genes in in Jrt Jrt mice mice contained contained genes genes that that are are known known toto be be important important for for the the osteoblast osteoblast function function (e.g., (e.g., BglapBglap——thethe gene gene coding coding for for osteocalcin; osteocalcin; Ifitm5Ifitm5——codingcoding for for BRIL) BRIL) and and the the synthesis synthesis of of extracellular extracellular matrix matrix (Smpd3 (Smpd3, ,Col11a1Col11a1, ,Col11a2Col11a2 andand Adamts14Adamts14) )(Table (Table 11).). TableTable 1. 1. ListList of of the the top top 20 20 upregulated upregulated genes in Jrt mice compared toto WTWT mice.mice. pp valuesvalues areare adjustedad- justedby the by Benjamini–Hochberg the Benjamini–Hochberg procedure, procedure, as determined as determined by deseq2. by deseq2. Fold GeneGene DescriptionDescription Fold Change pp Change Cyp2e1 Cytochrome P450, family 2, subfamily e, polypeptide 1 4.25 0.009 Cyp2e1 Cytochrome P450, family 2, subfamily e, polypeptide 1 4.25 0.009 BglapBglap BoneBone gamma gamma carboxyglutamate carboxyglutamate protein protein 3.443.44 <0.001<0.001 Bglap2Bglap2 BoneBone gamma-carboxyglutamate gamma-carboxyglutamate protein protein 2 2 3.143.14 <0.001<0.001 Cyp2f2Cyp2f2 CytochromeCytochrome P450, P450, family family 2, subfamily 2, subfamily f, polypeptide f, polypeptide 2 2 2.902.90 <0.001<0.001 Ighg2c 2.87 0.02 Ighg2c 2.87 0.02 Slc13a5 Solute carrier family 13, member 5 2.59 <0.001 Slc13a5Creb3l3 cAMPSolute responsive carrier family element 13, memberbinding protein 5 3-like 3 2.592.55 <0.0010.003 Creb3l3Tpsb2 cAMP responsive elementTryptase binding beta protein 2 3-like 3 2.552.38 0.0030.003 Col11a1Tpsb2 TryptaseCollagen, beta type 2 XI, alpha 1 2.382.34 0.0030.006 Col11a2 Collagen, type XI, alpha 2 2.31 0.015 Col11a1 Collagen, type XI, alpha 1 2.34 0.006 Lipc Lipase, hepatic 2.30 <0.001 Col11a2 Collagen, type XI, alpha 2 2.31 0.015 Lipc Lipase, hepatic 2.30 <0.001 Int. J. Mol. Sci. 2021, 22, 5290 3 of 12 Table 1. Cont. Gene Description Fold Change p Cgref1 Cell growth regulator with EF hand domain 1 2.29 <0.001 Ifi27l2a Interferon, alpha-inducible protein 27 like 2A 2.24 <0.001 Smpd3 Sphingomyelin phosphodiesterase 3, neutral 2.22 <0.001 Ifitm5 Interferon induced transmembrane protein 5 2.19 <0.001 Ctsw Cathepsin W 2.14 0.04 Kazald1 Kazal-type serine peptidase inhibitor domain 1 2.14 <0.001 Cthrc1 Collagen triple helix repeat containing 1 2.05 <0.001 Rerg RAS-like, estrogen-regulated, growth-inhibitor 2.05 0.002 A disintegrin-like and metallopeptidase with Adamts14 1.98 <0.001 thrombospondin type 1 motif, 14 In oim mice, the list of the top 20 upregulated genes also contained genes that play a role in the function of osteoblasts (e.g., Edn1, Ddit4, Cdkn1a, Igfbp3 and Stc2) (Table2). Only one gene (Cyp2e1) was among the top 20 upregulated genes in both Jrt and oim mice.