TBX6相關(guān)性先天性脊柱側(cè)凸臨床表型及致病機(jī)制研究
發(fā)布時(shí)間：2018-09-17 08:21
【摘要】：研究背景：先天性脊柱側(cè)凸(Congenital scoliosis, CS)是由胚胎期椎體發(fā)育異常導(dǎo)致的脊柱側(cè)方彎曲≥10°。CS具有進(jìn)展快、畸形重、并發(fā)癥多等特點(diǎn),嚴(yán)重時(shí)可致患者癱瘓,是造成青少年殘疾的主要疾病之一,給家庭和社會(huì)帶來極大負(fù)擔(dān)。脊椎動(dòng)物的脊柱是由體前中胚層周期性形成的體節(jié)發(fā)育而來,這一過程叫做體節(jié)形成(somitogenesis),體節(jié)形成異常即可導(dǎo)致先天性椎體畸形。由于其確切病因不明,目前尚無有效的預(yù)測手段,治療仍以支具或手術(shù)治療來緩解病情發(fā)展為主,因此探尋其發(fā)病原因,對于從病因?qū)W領(lǐng)域控制發(fā)病率、早期診斷和早期干預(yù)這種嚴(yán)重致殘性脊柱畸形疾病、緩解家庭和社會(huì)負(fù)擔(dān)具有重要意義。CS可由基因異�；颦h(huán)境因素變化導(dǎo)致,隨著越來越多的遺傳學(xué)病因報(bào)道,遺傳因素在CS發(fā)病中起到的作用逐漸被人們重視。CS是一種復(fù)雜、多基因、多遺傳模式疾病。目前國際上對于CS的遺傳學(xué)研究主要集中于單基因或染色體水平改變致病的家系研究或病例報(bào)道,而在點(diǎn)突變和染色體突變之間存在大片空白,即拷貝數(shù)變異(Copy number variation, CNV) 。在前期研究中,我們采用Agilent的人類全基因組CGH芯片對20例CS散發(fā)病例進(jìn)行全基因組掃描,在兩個(gè)病例(10%)的16p11.2區(qū)域都發(fā)現(xiàn)了一個(gè)同樣大小de novo缺失。該區(qū)域的TBX6基因在人類體節(jié)形成中起重要作用,TBX6基因突變或者缺失會(huì)產(chǎn)生脊柱發(fā)育障礙等表型。研究目的：研究TBX6相關(guān)性CS的致病率、致病機(jī)制及臨床表型,并建立相應(yīng)動(dòng)物模型。研究方法：首先通過qPCR、靶向CGH芯片及TBX6基因全長測序?qū)?37例散發(fā)CS患者16p11.2缺失及TBX6基因突變進(jìn)行檢測,之后對篩選出的TBX6相關(guān)性CS與非TBX6相關(guān)性CS進(jìn)行表型分析及對比,明確TBX6相關(guān)性CS表型特征。進(jìn)一步通過CRISPR-Cas9構(gòu)建TBX6相關(guān)性CS的斑馬魚模型,驗(yàn)證其表型特征及致病機(jī)制。研究結(jié)果：在237名散發(fā)CS患者中共發(fā)現(xiàn)23例TBX6無效變異,包括17例16p11.2/TBX6缺失,5例TBX6基因移碼突變及1例TBX6基因無義突變,并證明rs2289292-rs3809624-rs3809627單倍型T-C-A是除TBX6無效變異外CS發(fā)病的必要條件。表型分析證實(shí)TBX6相關(guān)性CS患者均存在一個(gè)或一個(gè)以上節(jié)段半椎體/楔形椎畸形,椎體畸形以半椎體/楔形椎為主(82.4%),TBX6相關(guān)性CS主要累及節(jié)段為下胸段及上腰段(T7-L3,82.2%),平均累累及椎體數(shù)為2.61,傾向于累及更少的脊柱節(jié)段；在CS合并肋骨畸形比例上,TBX6相關(guān)性與非TBX6相關(guān)性CS間不存在顯著差異,但有更高比例的簡單型肋骨畸形(60.9%)；此外,TBX6相關(guān)性CS傾向于更低的椎管內(nèi)畸形發(fā)生率(8.7%)。在斑馬魚模型中,tbx6及tbx16根據(jù)不同程度的突變表現(xiàn)出呈梯度的椎體畸形。研究結(jié)論：16p11.2/TBX6缺失是散發(fā)CS的重要遺傳學(xué)病因,其致病機(jī)制可能為TBX6劑量不足。該病因能解釋約10%的CS。我們將16p11.2缺失/TBX6無效變異合并T-C-A亞效等位基因的先天性脊柱側(cè)凸定義為TBX6相關(guān)性先天性脊柱側(cè)凸(TBX6-associated congenital scoliosis),其在臨床表型上也具有自身特點(diǎn)。在斑馬魚模型中,致病機(jī)制與表型均符合TBX6相關(guān)性CS的劑量模型,但其具體機(jī)制仍待進(jìn)一步研究。
[Abstract]:BACKGROUND: Congenital scoliosis (CS) is a lateral curvature of the spine (> 10 degrees) caused by abnormal vertebral development during embryonic period. CS is characterized by rapid progress, severe deformities and multiple complications. It can cause paralysis in severe cases. It is one of the main diseases causing disability in adolescents and brings great burden to family and society. The spine of an animal develops from somatogenesis, a process called somatogenesis, which is periodically formed in the anterior mesoderm. Abnormal somatogenesis can lead to congenital vertebral deformities. It is of great significance for controlling the incidence of CS from the field of etiology, early diagnosis and early intervention to relieve the burden of family and society. CS is a complex, multi-gene, multi-genetic model disease. At present, the genetics of CS is mainly focused on single gene or chromosome level change pathogenic family studies or case reports, but there is a large gap between point mutation and chromosome mutation, namely Copy number variation. In the previous study, we used Agilent's human genome-wide CGH chip to scan the entire genome of 20 sporadic CS cases. In both cases (10%) of the 16p11.2 region, an identical deletion of de novo was found. The TBX6 gene in this region plays an important role in the formation of human somatic segments, and the TBX6 gene mutates or deletes. Objective: To study the pathogenicity, pathogenesis and clinical phenotype of TBX6-related CS, and to establish animal models. Methods: TBX6 gene mutation and 16p11.2 deletion were detected in 237 sporadic CS patients by qPCR, CGH chip and TBX6 gene full-length sequencing. TBX6-related CS and non-TBX6-related CS were selected for phenotypic analysis and comparison. TBX6-related CS phenotypic characteristics were clarified. A zebrafish model of TBX6-related CS was constructed by CRISPR-Cas9 to verify its phenotypic characteristics and pathogenic mechanism. 2/TBX6 deletion, 5 TBX6 gene shift mutation and 1 TBX6 gene nonsense mutation proved that rs2289292-rs3809624-rs3809627 haplotype T-C-A was a necessary condition for the onset of CS except for the invalid variant of TBX6. Phenotypic analysis confirmed that one or more segmental hemivertebra/wedge vertebral malformations were present in all TBX6-related CS patients. TBX6-related CS mainly involved lower thoracic and upper lumbar segments (T7-L3,82.2%). The average number of vertebrae involved was 2.61, which tended to involve fewer vertebral segments. There was no significant difference between TBX6-related CS and non-TBX6-related CS in the ratio of CS with rib deformity, but there was a higher proportion of simple rib deformity (60%). In zebrafish models, Tbx6 and tbx16 exhibit gradient vertebral malformations based on varying degrees of mutation. The study concludes that 16p11.2/TBX6 deletion is an important genetic cause of sporadic CS, and its pathogenesis may be due to the inadequate dose of TBX6. TBX6-associated congenital scoliosis (TBX6-associated congenital scoliosis) is defined as a congenital scoliosis associated with the deletion of 16p11.2/TBX6 invalid variant and T-C-A subtype. It also has its own characteristics in clinical phenotype. In zebrafish models, the pathogenesis and phenotype are consistent with TBX6-related CS. However, the specific mechanism remains to be further studied.
【學(xué)位授予單位】：北京協(xié)和醫(yī)學(xué)院
【學(xué)位級別】：博士
【學(xué)位授予年份】：2015
【分類號】：R687.3
本文編號：2245287
[Abstract]:BACKGROUND: Congenital scoliosis (CS) is a lateral curvature of the spine (> 10 degrees) caused by abnormal vertebral development during embryonic period. CS is characterized by rapid progress, severe deformities and multiple complications. It can cause paralysis in severe cases. It is one of the main diseases causing disability in adolescents and brings great burden to family and society. The spine of an animal develops from somatogenesis, a process called somatogenesis, which is periodically formed in the anterior mesoderm. Abnormal somatogenesis can lead to congenital vertebral deformities. It is of great significance for controlling the incidence of CS from the field of etiology, early diagnosis and early intervention to relieve the burden of family and society. CS is a complex, multi-gene, multi-genetic model disease. At present, the genetics of CS is mainly focused on single gene or chromosome level change pathogenic family studies or case reports, but there is a large gap between point mutation and chromosome mutation, namely Copy number variation. In the previous study, we used Agilent's human genome-wide CGH chip to scan the entire genome of 20 sporadic CS cases. In both cases (10%) of the 16p11.2 region, an identical deletion of de novo was found. The TBX6 gene in this region plays an important role in the formation of human somatic segments, and the TBX6 gene mutates or deletes. Objective: To study the pathogenicity, pathogenesis and clinical phenotype of TBX6-related CS, and to establish animal models. Methods: TBX6 gene mutation and 16p11.2 deletion were detected in 237 sporadic CS patients by qPCR, CGH chip and TBX6 gene full-length sequencing. TBX6-related CS and non-TBX6-related CS were selected for phenotypic analysis and comparison. TBX6-related CS phenotypic characteristics were clarified. A zebrafish model of TBX6-related CS was constructed by CRISPR-Cas9 to verify its phenotypic characteristics and pathogenic mechanism. 2/TBX6 deletion, 5 TBX6 gene shift mutation and 1 TBX6 gene nonsense mutation proved that rs2289292-rs3809624-rs3809627 haplotype T-C-A was a necessary condition for the onset of CS except for the invalid variant of TBX6. Phenotypic analysis confirmed that one or more segmental hemivertebra/wedge vertebral malformations were present in all TBX6-related CS patients. TBX6-related CS mainly involved lower thoracic and upper lumbar segments (T7-L3,82.2%). The average number of vertebrae involved was 2.61, which tended to involve fewer vertebral segments. There was no significant difference between TBX6-related CS and non-TBX6-related CS in the ratio of CS with rib deformity, but there was a higher proportion of simple rib deformity (60%). In zebrafish models, Tbx6 and tbx16 exhibit gradient vertebral malformations based on varying degrees of mutation. The study concludes that 16p11.2/TBX6 deletion is an important genetic cause of sporadic CS, and its pathogenesis may be due to the inadequate dose of TBX6. TBX6-associated congenital scoliosis (TBX6-associated congenital scoliosis) is defined as a congenital scoliosis associated with the deletion of 16p11.2/TBX6 invalid variant and T-C-A subtype. It also has its own characteristics in clinical phenotype. In zebrafish models, the pathogenesis and phenotype are consistent with TBX6-related CS. However, the specific mechanism remains to be further studied.
【學(xué)位授予單位】：北京協(xié)和醫(yī)學(xué)院
【學(xué)位級別】：博士
【學(xué)位授予年份】：2015
【分類號】：R687.3
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