Identification and characterization of a novel risk factor for MS

 描述(由申请人提供)： 在招募多发性硬化患者进行外周血单核细胞研究的过程中，发现了一个有5个兄弟姐妹诊断为多发性硬化症的家庭。多发性硬化症在美国的患病率约为1：1000，有5个兄弟姐妹患多发性硬化症的几率极低，尚未有报道。某些罕见肿瘤的共病导致对肿瘤抑制基因STK11的测序分析，并在内含子V中发现了突变。LKB1激酶的STK11编码与T细胞的细胞代谢和炎症反应、胶质细胞的炎症反应和少突胶质细胞的成熟有关。这导致了我们的假设，STK11基因突变导致LKB1表达降低，导致MS患者T细胞激活增加，这有助于MS表型的发展。由于LKB1在MS中的作用尚未确定，我们进一步假设LKB1的表达或活性的变化发生在MS的小鼠模型EAE过程中。如果是这样，那么增加LKB1的治疗可能具有治疗益处。在这个项目中，我们将确定INDEX家族的其他成员是否存在STK11基因的相同或相似突变；并检验从这些家族成员分离的PBMC是否降低或改变了LKB1 mRNA的表达，并增加了T细胞的激活。由于STK11突变本身不足以诱导MS类型表型，我们将进行完整的外显子和基因组测序，以确定与STK11突变一起可能导致MS的相关变异。我们将通过对3,000名MS患者(复发缓解和原发进展型)和匹配对照的DNA样本(包括从海湾战争时期服役的不同种族退伍军人获得的样本)的PCR分析，确定是否有任何新的变异在MS人群中增加。利用人类T细胞，我们将确定STK11突变是如何影响T细胞激活的；然后通过实验操作LKB1的表达来确定对T细胞激活的影响。在最初的研究中，我们发现减少星形胶质细胞的LKB1会增加他们的炎症反应，因此我们将在体外研究LKB1在星形胶质细胞中的特征，并确定操纵LKB1是否会改变星形胶质细胞的反应。在小鼠中，我们将确定LKB1在EAE过程中脑、脊髓和外周T细胞中的表达是否发生变化。使用LKB1的小鼠，我们将测试从T细胞或星形胶质细胞有条件地敲除LKB1是否会导致或加重EAE疾病。最后，我们将测试FDA批准的治疗糖尿病的药物二甲双胍能否在体外选择性地诱导LKB1缺陷的T细胞凋亡。阳性结果将证明LKB1在MS疾病发展中的新作用，并提示二甲双胍或相关药物可用于减少T细胞LKB1表达缺陷的MS患者的活化T细胞。

 DESCRIPTION (provided by applicant): During the course of recruiting MS patients for a study of PBMCs, a family with 5 siblings diagnosed with MS was identified. The prevalence of MS in the US is roughly 1:1000, the odds of having 5 siblings with MS is extremely low and has not been reported. The presence of comorbidity for certain rare tumors led to sequencing analysis of tumor suppressor gene STK11, and a mutation was identified in intron V. STK11 codes for the LKB1 kinase which has been implicated in regulation of cellular metabolism and inflammatory responses in T cells, of inflammatory responses in glial cells, and in oligodendrocyte maturation. This leads to our hypothesis that mutations in STK11 gene leading to a reduction in LKB1 expression cause increased activation of T cells in MS patients which contributes to development of an MS phenotype. Since the role of LKB1 in MS has not been characterized, we further hypothesize that changes in LKB1 expression or activity occur during the course of EAE, the mouse model of MS. If so, then treatments to increase LKB1 may be of therapeutic benefit. In this project, we will determine if other members of the index family harbor the same or similar mutation in the STK11 gene; and test the hypothesis that PBMCs isolated from these family members have reduced or altered expression of LKB1 mRNA, and increased T cell activation. Since a mutation in STK11 alone is not sufficient to induce an MS type phenotype we will carry out full exomic and genomic sequencing to identify associated variants which together with the STK11 mutation could lead to MS. We will determine if the prevalence of any novel variants are increased in the MS population by PCR analysis of DNA samples from 3,000 MS patients (both relapsing remitting and primary progressive forms) and matched controls, including samples obtained from military Veterans of different races who served in the Gulf War Era. Using human T cells, we will determine how the STK11 mutation influences T cell activation; then experimentally manipulate LKB1 expression to identify effects on Tcell activation. In initial studies we found tha reducing LKB1 from astrocytes increase their inflammatory responses, we will therefore characterize LKB1 in astrocyte in vitro and determine if manipulating LKB1 alters astrocyte responses. In mice, we will determine if expression of LKB1 changes during the course of EAE in brain, spinal cord, and in peripheral T cells. Using an LKB1 floxed mouse, we will test if conditional knockout of LKB1 from T cells or astrocytes leads to or exacerbates EAE disease. Finally, we will test if metformin, an FDA approved drug to treat diabetes, can selectively induce apoptosis in the LKB1 deficient T cells in vitro. Positive findings will demonstrate a novel role fr LKB1 in the development of MS disease, and suggest that metformin or related drugs could be used to reduce activated T cells in MS patients who have deficiencies in Tcell LKB1 expression.