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Summary Medullary thyroid carcinoma (MTC) develops from hyperplasia of thyroid C cells and represents one of the major causes of thyroid cancer mortality. Mutations in the cysteine‐rich domain (CRD) of the RET gene are the most prevalent genetic cause of MTC. The current consensus holds that such cysteine mutations cause ligand‐independent dimerization and constitutive activation of RET. However, given the number of the CRD mutations left uncharacterized, our understanding of the pathogenetic mechanisms by which CRD mutations lead to MTC remains incomplete. We report here that RET(C618F), a mutation identified in MTC patients, displays moderately high basal activity and requires the ligand for its full activation. To assess the biological significance of RET(C618F) in organogenesis, we generated a knock‐in mouse line conditionally expressing RET(C618F) cDNA by the Ret promoter. The RET(C618F) allele can be made to be Ret‐null and express mCherry by Cre‐loxP recombination, which allows the assessment of the biological influence of RET(C618F) in vivo. Mice expressing RET(C618F) display mild C cell hyperplasia and increased numbers of enteric neurons, indicating that RET(C618F) confers gain‐of‐function phenotypes. This mouse line serves as a novel biological platform for investigating pathogenetic mechanisms involved in MTC and enteric hyperganglionosis.

Missense mutations of the RET gene have been identified in both multiple endocrine neoplasia (MEN) type 2A/B and Hirschsprung disease (HSCR: congenital absence of the enteric nervous system, ENS). Current consensus holds that MEN2A/B and HSCR are caused by activating and inactivating RET mutations, respectively. However, the biological significance of RET missense mutations in vivo has not been fully elucidated. In the present study, we introduced one MEN2B-associated (M918T) and two HSCR-associated (N394K and Y791F) RET missense mutations into the corresponding regions of the mouse Ret gene by genome editing (Ret M919T , Ret N396K and Ret Y792F ) and performed histological examinations of Ret-expressing tissues to understand the pathogenetic impact of each mutant in vivo. Ret M919T/+ mice displayed MEN2B-related phenotypes, including C-cell hyperplasia and abnormal enlargement of the primary sympathetic ganglia. Similar sympathetic phenotype was observed in Ret M919T/mice, demonstrating a strong pathogenetic effect of the Ret M918T by a single-allele expression. In contrast, no abnormality was found in the ENS of mice harboring the Ret N394K or Y791F mutation. Most surprisingly, single-allele expression of RET N394K or Y791F was sufficient for normal ENS development, indicating that these RET mutants exert largely physiological function in vivo. This study reveals contrasting pathogenetic effects between MEN2B-and HSCR-associated RET missense mutations, and suggests that some of HSCR-associated RET missense mutations are by themselves neither inactivating nor pathogenetic and require involvement of other gene mutations for disease expressivity. K E Y W O R D S gene mutation, genome editing, Hirschsprung disease, multiple endocrine neoplasia, RET | 215 NAKATANI eT Al.

Intermittent parathyroid hormone (PTH) administration is known to promote bone healing after surgical procedures. However, the mechanism and influence of PTH on the mineral and collagen quality of the jaw are not well understood. Most studies have focused on analyzing the bone density and microstructure of the mandible, and have insufficiently investigated its mineral and collagen quality. Oxidative stress activates osteoclasts, produces advanced glycation end products, and worsens mineral and collagen quality. We hypothesized that PTH induces oxidation and affects the mineral and collagen quality of newly formed mandibular bone. To test this, we examined the mineral and collagen quality of newly formed mandibular bone in rats administered PTH, and analyzed serum after intermittent PTH administration to examine the degree of oxidation. PTH administration reduced mineralization and worsened mineral and collagen quality in newly formed bone. In addition, total anti-oxidant capacity in serum was significantly decreased and the oxidative-INDEX was increased among PTH-treated compared to vehicle-treated rats, indicating serum oxidation. In conclusion, intermittent administration of PTH reduced mineral and collagen quality in newly formed mandibular bone. This effect may have been induced by oxidation.

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Bone quality is defined by structural and material characteristics. Most studies on the mandible have focused on the analysis of structural characteristics, with insufficient investigation of material characteristics. This study tested whether zoledronate affects the material characteristics of newly formed mandibular bone. Thirty-six female Wistar rats were assigned to three groups: sham-ovariectomized rats (SHAM, n=12), ovariectomized rats (OVX, n=12), and ovariectomized rats treated with zoledronate (ZOL, n=12). The left side of the mandibular ramus of all rats was drilled bicortically. Twenty-eight days after surgery, all surviving rats were euthanized and all mandibles were removed. Raman microspectroscopy was performed, and five spectra per specimen of newly formed mandibular bone were analysed. Compared with OVX rats, the mineral/matrix ratio in ZOL rats was significantly increased (5.43±1.88 vs. 7.86±2.05), while crystallinity (0.055±0.002 vs. 0.050±0.002), relative proteoglycan content (0.43±0.10 vs. 0.31±0.05), and collagen structural integrity (1.16±0.21 vs. 0.72±0.06) were significantly decreased. These changes in material characteristics may explain why rats that received zoledronate exhibited peculiar biological phenomena such as bisphosphonate-related osteonecrosis of the jaw.