Individuals with neurofibromatosis type 1 (NF1) have a high incidence of osteoporosis and osteopenia. However, understanding of the cellular and molecular basis of these sequelae is incomplete. Osteoclasts are specialized myeloid cells that are the principal bone-resorbing cells of the skeleton. We found that Nf1 +/-mice contain elevated numbers of multinucleated osteoclasts. Both osteoclasts and osteoclast progenitors from Nf1 +/-mice were hyperresponsive to limiting concentrations of M-CSF and receptor activator of NF-κB ligand (RANKL) levels. M-CSF-stimulated p21 ras -GTP and Akt phosphorylation was elevated in Nf1 +/-osteoclasts associated with gains of function in survival, proliferation, migration, adhesion, and lytic activity. These gains of function are associated with more severe bone loss following ovariectomy as compared with that in syngeneic WT mice. Intercrossing Nf1 +/-mice and mice deficient in class 1 A PI3K (p85α) restored elevated PI3K activity and Nf1 +/-osteoclast functions to WT levels. Furthermore, in vitro-differentiated osteoclasts from NF1 patients also displayed elevated Ras/PI3K activity and increased lytic activity analogous to those in murine Nf1 +/-osteoclasts. Collectively, our results identify a what we believe to be a novel cellular and biochemical NF1-haploinsufficient phenotype in osteoclasts that has potential implications for the pathogenesis of NF1 bone disease. IntroductionNeurofibromatosis type 1 (NF1) is a common, pandemic genetic disorder that is characterized by a range of both malignant and nonmalignant manifestations and is caused by mutations in the NF1 gene. Neurofibromin, the protein encoded by NF1, functions as a GTPase-activating protein (GAP) for Ras. Though loss of both alleles of NF1 in tumorigenic cells is consistent with NF1 being a tumor suppressor gene, recent genetic evidence in murine models has indicated that nullizygous loss of Nf1 in the tumorigenic cells of plexiform neurofibromas (1) and optic gliomas (2) is necessary but not sufficient for tumor progression, though tumors occur with high penetrance when lineages of the tumor microenvironment are haploinsufficient at Nf1. These in vivo data provide evidence that haploinsufficiency of Nf1 in at least a subset of lineages has a role in the malignant manifestations of NF1 and also suggest a potential role for haploinsufficiency of Nf1 (NF1) in the nonmalignant pathogenesis of NF1.Three recent reports, including a controlled trial using WHO criteria for osteoporosis and osteopenia, have provided evidence that NF1 patients have a significantly higher incidence of osteoporosis and osteopenia (3-5). Bone homeostasis is maintained by balancing skeletal matrix formation and resorption. Osteoclasts are specialized cells derived from the myeloid monocyte/macro-

Inactivating mutations in chromodomain helicase DNA binding protein 7 (CHD7) cause CHARGE syndrome, a severe multiorgan system disorder of which Isolated gonadotropin-releasing hormone (GnRH) deficiency (IGD) is a minor feature. Recent reports have described predominantly missense CHD7 alleles in IGD patients, but it is unclear if these alleles are relevant to causality or overall genetic burden of Kallmann syndrome (KS) and normosmic form of IGD. To address this question, we sequenced CHD7 in 783 well-phenotyped IGD patients lacking full CHARGE features; we identified nonsynonymous rare sequence variants in 5.2% of the IGD cohort (73% missense and 27% splice variants). Functional analyses in zebrafish using a surrogate otolith assay of a representative set of these CHD7 alleles showed that rare sequence variants observed in controls showed no altered function. In contrast, 75% of the IGD-associated alleles were deleterious and resulted in both KS and normosmic IGD. In two families, pathogenic mutations in CHD7 coexisted with mutations in other known IGD genes. Taken together, our data suggest that rare deleterious CHD7 alleles contribute to the mutational burden of patients with both KS and normosmic forms of IGD in the absence of full CHARGE syndrome. These findings (i) implicate a unique role or preferential sensitivity for CHD7 in the ontogeny of GnRH neurons, (ii) reiterate the emerging genetic complexity of this family of IGD disorders, and (iii) demonstrate how the coordinated use of well-phenotyped cohorts, families, and functional studies can inform genetic architecture and provide insights into the developmental biology of cellular systems.CHD7 | Kallmann syndrome | idiopathic hypogonadotropic hypogondism | CHARGE syndrome | missense mutations

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