ENPP1 enzyme replacement therapy improves ectopic calcification but does not rescue skeletal phenotype in a mouse model for craniometaphyseal dysplasia.

Craniometaphyseal dysplasia (CMD) is a rare genetic bone disorder, characterized by progressive thickening of craniofacial bones and flared metaphyses of long bones. Craniofacial hyperostosis leads to the obstruction of neural foramina and neurological symptoms such as facial palsy, blindness, deafness, or severe headache. Mutations in ANKH (mouse ortholog ANK), a transporter of small molecules such as citrate and ATP, are responsible for autosomal dominant CMD. Knock-in (KI) mice carrying an ANKF377del mutation (AnkKI/KI ) replicate many features of human CMD. Pyrophosphate (PPi) levels in plasma are significantly reduced in AnkKI/KI mice. PPi is a potent inhibitor of mineralization. To examine the extent to which restoration of circulating PPi levels may prevent the development of a CMD-like phenotype, we treated AnkKI/KI mice with the recombinant human ENPP1-Fc protein IMA2a. ENPP1 hydrolyzes ATP into AMP and PPi. Male and female Ank+/+ and AnkKI/KI mice (n ≥ 6/group) were subcutaneously injected with IMA2a or vehicle weekly for 12 wk, starting at the age of 1 wk. Plasma ENPP1 activity significantly increased in AnkKI/KI mice injected with IMA2a (Vehicle/IMA2a: 28.15 ± 1.65/482.7 ± 331.2 mOD/min; p <.01), which resulted in the successful restoration of plasma PPi levels (Ank+/+ /AnkKI/KI vehicle treatment/AnkKI/KI IMA2a: 0.94 ± 0.5/0.43 ± 0.2/1.29 ± 0.8 µM; p <.01). We examined the skeletal phenotype by X-Ray imaging and µCT. IMA2a treatment of AnkKI/KI mice did not significantly correct CMD features such as the abnormal shape of femurs, increased bone mass of mandibles, hyperostotic craniofacial bones, or the narrowed foramen magnum. However, µCT imaging showed ectopic calcification near basioccipital bones at the level of the foramen magnum and on joints of AnkKI/KI mice. Interestingly, IMA2a treatment significantly reduced the volume of calcified nodules at both sites. Our data demonstrate that IMA2a is sufficient to restore plasma PPi levels and reduce ectopic calcification but fails to rescue skeletal abnormalities in AnkKI/KI mice under our treatment conditions.

Loss-of-function OGFRL1 variants identified in autosomal recessive cherubism families.

Cherubism (OMIM 118400) is a rare craniofacial disorder in children characterized by destructive jawbone expansion due to the growth of inflammatory fibrous lesions. Our previous studies have shown that gain-of-function mutations in SH3 domain-binding protein 2 (SH3BP2) are responsible for cherubism and that a knock-in mouse model for cherubism recapitulates the features of cherubism, such as increased osteoclast formation and jawbone destruction. To date, SH3BP2 is the only gene identified to be responsible for cherubism. Since not all patients clinically diagnosed with cherubism had mutations in SH3BP2, we hypothesized that there may be novel cherubism genes and that these genes may play a role in jawbone homeostasis. Here, using whole exome sequencing, we identified homozygous loss-of-function variants in the opioid growth factor receptor like 1 (OGFRL1) gene in 2 independent autosomal recessive cherubism families from Syria and India. The newly identified pathogenic homozygous variants were not reported in any variant databases, suggesting that OGFRL1 is a novel gene responsible for cherubism. Single cell analysis of mouse jawbone tissue revealed that Ogfrl1 is highly expressed in myeloid lineage cells. We generated OGFRL1 knockout mice and mice carrying the Syrian frameshift mutation to understand the in vivo role of OGFRL1. However, neither mouse model recapitulated human cherubism or the phenotypes exhibited by SH3BP2 cherubism mice under physiological and periodontitis conditions. Unlike bone marrow-derived M-CSF-dependent macrophages (BMMs) carrying the SH3BP2 cherubism mutation, BMMs lacking OGFRL1 or carrying the Syrian mutation showed no difference in TNF-ɑ mRNA induction by LPS or TNF-ɑ compared to WT BMMs. Osteoclast formation induced by RANKL was also comparable. These results suggest that the loss-of-function effects of OGFRL1 in humans differ from those in mice and highlight the fact that mice are not always an ideal model for studying rare craniofacial bone disorders.

Skeletal abnormalities caused by a Connexin43R239Q mutation in a mouse model for autosomal recessive craniometaphyseal dysplasia.

Craniometaphyseal dysplasia (CMD), a rare craniotubular disorder, occurs in an autosomal dominant (AD) or autosomal recessive (AR) form. CMD is characterized by hyperostosis of craniofacial bones and flaring metaphyses of long bones. Many patients with CMD suffer from neurological symptoms. To date, the pathogenesis of CMD is not fully understood. Treatment is limited to decompression surgery. Here, we report a knock in (KI) mouse model for AR CMD carrying a R239Q mutation in CX43. Cx43KI/KI mice replicate many features of AR CMD in craniofacial and long bones. In contrast to Cx43+/+ littermates, Cx43KI/KI mice exhibit periosteal bone deposition and increased osteoclast (OC) numbers in the endosteum of long bones, leading to an expanded bone marrow cavity and increased cortical bone thickness. Although formation of Cx43+/+ and Cx43KI/KI resting OCs are comparable, on bone chips the actively resorbing Cx43KI/KI OCs resorb less bone. Cortical bones of Cx43KI/KI mice have an increase in degenerating osteocytes and empty lacunae. Osteocyte dendrite formation is decreased with reduced expression levels of Fgf23, Sost, Tnf-α, IL-1ß, Esr1, Esr2, and a lower Rankl/Opg ratio. Female Cx43KI/KI mice display a more severe phenotype. Sexual dimorphism in bone becomes more evident as mice age. Our data show that the CX43R239Q mutation results in mislocalization of CX43 protein and impairment of gap junction and hemichannel activity. Different from CX43 ablation mouse models, the CX43R239Q mutation leads to the AR CMD-like phenotype in Cx43KI/KI mice not only by loss-of-function but also via a not yet revealed dominant function.