基于骨肉瘤近亲婚配家系的外显子组测序鉴定骨肉瘤的易感基因

Osteosarcoma is the most representative tumor of malignant bone neoplasm with high morbidity and mortality. Current treatment effect is not satisfied. Previous studies have revealed that patients with Li Fraumeni syndrome and familial retinoblastoma were tend to have osteosarcoma. Furthermore, although the most cases are sporadic without a known genetic or environmental cause, familial cases were observed for osteosarcomas, suggesting genetic factors play a role in the development of this disease. One research direction at present is to explore the genetic etiology of osteosarcoma, and then provide new ways for the treatment. Consanguineous families are useful for mapping human genes that cause recessive traits. In this study, we recruited a consanguineous family, including two probands with osteosarcoma and their unaffected parents. By exome sequencing on the genomic DNA of members of the consanguineous family, we found both of the two patients with osteosarcoma were homozygous for a nonsynonymous rare variant p.T897I on GENE1 at chromosome 8, while their parents were heterozygous for the variant. However, there is no published study on the biological function and molecular mechanisms of GENE1 in tumorigenesis of osteosarcoma. In this proposal, The functional rare variants in GENE1 will be screened in an case-control population of osteosarcoma. The biological function of GENE1 in osteosarcoma will be investigated in multiple osteosarcoma cell lines (in vitro), and in human osteosarcoma xenograft transplanted nude mice with subcutaneous injection (in vivo). Furthermore, Expression profiling and IP-MASS analysis will be used to search for proteins interacted with and signal pathways regulated by this gene in osteosarcoma. q-PCR and immunohistochemistry will be used to detect mRNA and protein level of GENE1 in osteosarcoma tissues and paired adjacent tissues. Finally, the relations between GENE1 expression and clinical parameters of patients with osteosarcoma will be evaluated. The present study on GENE1 will provide experimental basis for understanding the genetic and functional role of this gene in osteosarcoma.

骨肉瘤是最常见的恶性骨肿瘤，目前治疗效果仍不尽人意。以往的研究显示，有诸多遗传综合征患者易继发骨肉瘤，提示其发病与遗传密切相关。鉴定骨肉瘤的遗传学病因，可为其治疗提供新的思路。近亲婚配家系可用于定位遵循隐性遗传模式疾病的致病基因。我们招募了一个骨肉瘤的近亲婚配家系，包括两位亲兄妹骨肉瘤患者，其父母为近亲结婚。通过对该家系进行高通量外显子组测序，发现在8号染色体上的GENE1上存在一个隐性纯合的非同义变异（p.T897I），即该变异在两位患者中均为纯合子，而在父母中均为杂合子。目前尚未见GENE1与骨肉瘤的报道，但我们初步的实验表明敲低GENE1的表达能够显著促进骨肉瘤细胞系的生长。本项目拟基于前期研究基础上，在分子、细胞、动物以及临床样本多个层次，对我们自主发现的候选基因开展遗传学、功能和机制研究，希望该研究能有助于揭示骨肉瘤的发病机制，并为骨肉瘤的防诊治提供新的候选生物标志物和药物靶标。

骨肉瘤是最常见的恶性骨肿瘤，目前治疗效果仍不尽人意。以往的研究显示，有诸多遗传综合征患者易继发骨肉瘤，提示其发病与遗传密切相关。鉴定骨肉瘤的遗传学病因，可为其治疗提供新的思路。为此，我们招募了一个骨肉瘤的近亲婚配家系，包括两位亲兄妹骨肉瘤患者，其父母为近亲结婚，然后通过高通量外显子组测序来定位骨肉瘤的致病基因。我们发现在8号染色体上的GENE1上存在一个隐性纯合的非同义罕见胚系变异（p.T897I），即该变异在两位患者中均为纯合子，而在父母中均为杂合子。功能研究表明，GENE1基因能够促进细胞凋亡、抑制细胞增殖，而GENE1 T897I突变体则抑制细胞凋亡、促进细胞增殖，提示GENE1 T897I突变在功能上可能能够导致骨肉瘤发病。蛋白-蛋白相互作用分析发现，GENE1的相互作用分子可能有ATM、ATR、P53、SIRT1、BRD2、XRCC1等，其中有部分是以往没有报道的新的候选互作分子。基因通路富集分析显示，GENE1的表达变化与P53等通路有关。进一步的人群验证研究发现，与对照相比（4/1961），GENE1非同义罕见胚系变异在骨肉瘤患者（2/74）中的比例显著增加（P=0.019; OR=13.19, 95%CI:1.18-93.79）；此外，BRD2非同义罕见胚系变异（p.E726D）携带者的肿瘤发病风险显著增加（P=0.0014），其中主要是肺癌和结直肠癌发病风险显著增加（P值分别为0.0072和2.8×10-5）。.上述研究结果提示GENE1、BRD2分别是骨肉瘤以及肺癌、结直肠癌的新型候选易感基因，携带GENE1、BRD2罕见胚系变异可能分别是导致骨肉瘤以及肺癌、结直肠癌发病风险增加的重要因素。有必要进一步在独立的病例对照人群中开展重复验证。研究结果申请了中国发明专利一项（申请号：202010998128.7，图11），发表SCI论文一篇（Cuili Shu, Cheng Liu. Liquid nitrogen for cryotherapy treatment for osteosarcoma of the middle femur: A case report. Journal of Clinical Laboratory Analysis, Accepted, 2020/12/30. DOI:10.1002/jcla.23701.出版中。