A study on the effect of Wnt16 on cortical bone quality in adolescent idiopathic scoliosis

M.Phil.IntroductionAdolescent Idiopathic Scoliosis (AIS) is a three-dimensional (3D) spinal deformity that occurs during pubertal growth spurt with multifactorial pathomechanisms underlying the initiation phase and progression phase. Collective evidence on the association between AIS and abnormal skeletal growth with systemic low bone mass has been well documented. However, the underlying mechanism for systemic low bone mass in AIS remains unclear. Wnt signalling pathway is one of the important pathways regulating bone metabolism and influencing bone strength. Recent GWAS studies have shown Wnt family ligand 16 (WNT16) associates with reduced cortical bone mineral density, structure and thus strength. Studies with transgenic mice with the deletion of Wnt16 have demonstrated abnormal cortical bone qualities. A randomized double-blinded placebo-controlled trial conducted by our team has indicated that VitD supplementation could improve BMD and concurrently prevent curve progression in AIS girls with low bone mass.HypothesisWe hypothesized that WNT16 genetic variation and circulating bone markers could affect bone accrual during the rapid growth phase, leading to decreased cortical bone thickness and vBMD in AIS. Although WNT16 could be a potential therapeutic target, there is lack of understanding on what can help in modulating its signal. With our RCT study showing that VitD supplementation could improve bone quality in AIS with low BMD clinically in a dosage dependent manner, it was also aimed to evaluate whether VitD could ameliorate the abnormal cortical bone phenotypes.Objectives1. To determine the genotype frequencies of selected SNPs among patients with AIS Vs healthy controls and to determine the associations between BMD/fracture related gene polymorphisms and bone qualities in patients with AIS, and its association with curve severity in AIS.2. To compare the circulating bone markers between AIS and age- and gender- matched healthy control and to evaluate the relationship between the tested bone markers and bone qualities in AIS and healthy control.3. To determine the cortical bone quality at femoral mid-shaft of Wnt16 gKO mice Vs homozygous Wnt16+/+ as control at age of 4, 7 and 10 weeks. To study if deficient, sufficient and high dose dietary VitD would further affect the cortical bone quality in Wnt16 gKO mice Vs homozygous Wnt16+/+ during rapid growth phase.Key findings1. Associations between Wnt16 genetic polymorphism and bone qualities in AIS and healthy control were not apparent. However, BMD related gene polymorphisms were found to be associated with bone qualities in healthy adolescents but not in AIS.2. AIS participants demonstrated significantly lower plasma level of WNT16 and elevated serum level of type I procollagen N-terminal propeptide (P1NP) and sclerostin. Healthy control showed significant association between circulating bone markers and bone qualities which was not observed in AIS, indicating the likelihood of imbalanced bone metabolism in AIS.3. Wnt16 global knock out (gKO) mice demonstrated lower cortical bone density compared with WT mice from age of 4 weeks to 10 weeks. WT mice with sufficient dietary VitD were found to be favourable to cortical bone accrual, whereas deficient or high dosage of dietary VitD resulted in poorer cortical bone qualities at the age of 10 weeks old. Wnt16 gKO mice were less prone to cortical bone loss induced by high dosage of dietary VitD.DiscussionGenetic studies did not demonstrate association between BMD related genetic polymorphism and bone qualities in AIS, indicating environmental factors may contributing more to the systemic low bone mass in AIS. To the best of our knowledge, this is the first study demonstrating the reduced plasma level of WNT16 in AIS vs healthy control participants and indicating the possible role of WNT16 in regulating abnormal cortical bone mass and structure in AIS. Wnt16 gKO mice demonstrated lower cortical bone vBMD, thickness and bone volume ratio when compared with WT mice, which supported its biological role in cortical bone accrual. In WT mice, sufficient dietary VitD seems to be adequate dosage to render beneficiary effect on cortical bone accrual in contrast to the negative effect observed in deficient and high dose dietary groups. On the other hand, Wnt16 gKO mice appeared to be less vulnerable to cortical bone loss upon high dosage of dietary VitD treatment.Further validation studies on the biological role of Wnt16 and crosstalk with VitD metabolism on cortical and trabecular bone qualities is warranted for further investigation with conditional knockout transgenic animal models and with molecular function studies in cellular models, such as osteoblast and osteoclast to facilitate comprehensive understanding in causal relationship between imbalanced bone cells activities and derange bone qualities in patients with AIS.前言青少年特發性脊柱側彎(AIS)是發生在青春期骨骼快速發育階段的3D脊柱畸形，其發生與進展與多種基因及環境種因素有關。業已證實，AIS和骨骼發育異常與骨質密度減低具有相關性。然而，低骨質密度導致AIS發生與進展的機制尚未明確。Wnt信號通路是調節骨骼代謝、影響骨骼強度的重要通路之一。其中，Wnt16與骨骼強度減少、骨骼品質具有顯著的相關性。本課題組的前期隨機對照研究結果提示，維生素D與鈣聯合補充劑(每日600 mg鈣劑與800 IU維生素D3)能夠提高AIS患者的骨質密度、預防脊柱側彎嚴重程度的進展。研究假設我們假設Wnt16變異類型及其血液標記物異常可能影響AIS患者快速發育階段的骨骼發育水準，且該影響將導致較低的皮質骨厚度和骨質密度。維生素D補充劑能夠減輕該影響導致的骨骼品質異常。研究目標1. 探究AIS患者與健康對照組受試者體內單核苷酸多態性的基因頻率，並探索AIS患者骨質密度/骨折相關基因多態性與骨骼品質及側彎嚴重程度之間的相關性。2. 對比AIS與年齡、性別匹配對照組受試者的血液骨轉換生化標記物水準，並探討AIS患者組及對照組該標記物水準與骨骼品質之間的相關性。3. 對比Wnt16 基因敲除鼠與野生型小鼠於4、7、10周齡時股骨中段皮質骨骨骼品質，並探究維生素D缺乏、高劑量維生素D飲食、正常飲食對Wnt16 基因敲除鼠及野生型鼠快速生長期皮質骨骨骼品質的影響。主要結果1. AIS患者組及健康對照組中，WNT16基因多態性及骨骼品質無顯著相關性。在兩組受試者中，骨骼品質相關基因多態性與骨骼品質具有顯著相關性。2. 對比健康對照組，AIS患者組血液WNT16水準較低，P1NP與人骨硬化蛋白水準較高，差異具有統計學意義。健康對照組中，骨轉換生化標記物水準和骨骼品質具有顯著相關性，但該相關性在AIS患者組中並不具有統計學意義。本結果提示AIS患者可能存在骨代謝水準失衡。3. 相比同齡野生型小鼠，4、7、10周齡Wnt16基因敲除鼠具有較低的皮質骨密度。4. 在野生型小鼠中，相較于正常飲食組，維生素D缺乏及高劑量維生素D飲食組10周齡小鼠皮質骨品質較低。5. 相較於野生型小鼠，Wnt16基因敲除鼠的高劑量維生素D飲食所致皮質骨密度丟失較少。討論本實驗中，有關結果並未證實AIS患者中骨骼品質相關基因多態性和骨骼品質具有顯著的相關性。該結果提示環境因素可能是導致AIS患者低骨骼品質的主要方面。本研究首次提出，AIS患者具有較低的血液WNT16水準，提示WNT16可能具有調節AIS患者皮質骨品質和結構的功能。Wnt16基因全敲除鼠相較於野生型小鼠具有較低的皮質骨體積骨質密度、皮質骨厚度以及皮質骨骨骼體積分數，提示Wnt16在皮質骨骨骼生長中具有重要作用。本研究亦提示正常維生素D含量的飲食有助於皮質骨的生長，但維生素D缺乏及高劑量維生素D飲食組均具有較差的皮質骨品質。Wnt16基因敲除鼠的高劑量維生素D飲食所致皮質骨密度丟失較少。以上研究結果證實：1）Wnt16具有調節皮質骨骨量的生物學作用；2）維生素D可能具有改善Wnt16 基因全敲除所致骨骼品質減少的作用。進一步研究將著眼於在Wnt16特異性敲除小鼠中驗證Wnt16之於皮質骨骨骼品質發育的生物學功能，並通過基因過表達和基因敲除的細胞學功能驗證。亦需進一步探索成骨細胞、破骨細胞內Wnt16 與維生素D的交互作用機制及信號通路，為更加全面、深入地闡述骨骼細胞活動和骨骼品質失衡之間的因果關係提供理論基礎。Cheng, Ka Lo.Thesis M.Phil. Chinese University of Hong Kong 2020.Includes bibliographical references (leaves 109-125).Abstracts also in Chinese.Title from PDF title page (viewed on March 17, 2022)